Metal chelate containing compositions for use in chemiluminescent assays

ABSTRACT

Compositions are disclosed comprising (a) a metal chelate wherein the metal is selected from the group consisting of europium, terbium, dysprosium, samarium osmium and ruthenium in at least a hexacoordinated state and (b) a compound having a double bond substituted with two aryl groups, an oxygen atom and an atom selected from the group consisting of oxygen, sulfur and nitrogen wherein one of the aryl groups is electron donating with respect to the other. Such composition is preferably incorporated in a latex particulate material. Methods and kits are also disclosed for determining an analyte in a medium suspected of containing the analyte. The methods and kits employ as one component a composition as described above.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of co-pending patentapplication U.S. Ser. No. 07/704,569 filed May 22, 1991, entitled “AssayMethod Utilizing Induced Luminescence,” the disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates to methods, compositions and kits fordetermining an analyte in a sample. In particular, this inventionrelates to compositions that exhibit a high quantum yieldchemiluminescence when activated by singlet oxygen, decay rapidly andemit at long wavelengths.

[0004] The clinical diagnostic field has seen a broad expansion inrecent years, both as to the variety of materials (analytes) that may bereadily and accurately determined, as well as the methods for thedetermination. Convenient, reliable and non-hazardous means fordetecting the presence of low concentrations of materials in liquids isdesired. In clinical chemistry these materials may be present in bodyfluids in concentrations below 10⁻¹² molar. The difficulty of detectinglow concentrations of these materials is enhanced by the relativelysmall sample sizes that can be utilized.

[0005] In developing an assay there are many considerations. Oneconsideration is the signal response to changes in the concentration ofanalyte. A second consideration is the ease with which the protocol forthe assay may be carried out. A third consideration is the variation ininterference from sample to sample. Ease of preparation and purificationof the reagents, availability of equipment, ease of automation andinteraction with material of interest are some of the additionalconsiderations in developing a useful assay.

[0006] One broad category of techniques involves the use of a receptorwhich can specifically bind to a particular spacial and polarorganization of a labeled ligand as a function of the presence of ananalyte. The observed effect of binding by the receptor will depend uponthe label. In some instances the binding of the receptor merely providesfor a differentiation in molecular weight between bound and unboundlabeled ligand. In other instances the binding of the receptor willfacilitate separation of bound labeled ligand from free labeled ligandor it may affect the nature of the signal obtained from the label sothat the signal varies with the amount of receptor bound to labeledligand. A further variation is that the receptor is labeled and theligand unlabeled. Alternatively, both the receptor and ligand arelabeled or different receptors are labeled with two different labels,whereupon the labels interact when in close proximity and the amount ofligand present affects the degree to which the labels of the receptormay interact.

[0007] There is a continuing need for new and accurate techniques thatcan be adapted for a wide spectrum of different ligands or be used inspecific cases where other methods may not be readily adaptable.

[0008] Homogeneous immunoassays have previously been described for smallmolecules. These assays include SYVA's FRAT® assay, EMIT® assay, enzymechanneling immunoassay, and fluorescence energy transfer immunoassay(FETI); enzyme inhibitor immunoassays (Hoffman LaRoche and AbbottLaboratories): fluorescence polarization immunoassay (Dandlicker), amongothers. All of these methods have limited sensitivity, and only a fewincluding FETI and enzyme channeling, are suitable for largemultiepitopic analytes. Luminescent compounds, such as fluorescentcompounds and chemiluminescent compounds, find wide application in theassay field because of their ability to emit light. For this reason,luminescers have been utilized as labels in assays such as nucleic acidassays and immunoassays. For example, a member of a specific bindingpair is conjugated to a luminescer and various protocols are employed.The luminescer conjugate can be partitioned between a solid phase and aliquid phase in relation to the amount of analyte in a sample suspectedof containing the analyte. By measuring the luminescence of either ofthe phases, one can relate the level of luminescence observed to aconcentration of the analyte in the sample.

[0009] Particles, such as liposomes and erythrocyte ghosts, have beenutilized as carriers of encapsulated water soluble materials. Forexample, liposomes have been employed to encapsulate biologically activematerial for a variety of uses, such as drug delivery systems wherein amedicament is entrapped during liposome preparation and thenadministered to the patient to be treated.

[0010] Particles, such as latex beads and liposomes, have also beenutilized in assays. For example, in homogeneous assays an enzyme may beentrapped in the aqueous phase of a liposome labelled with an antibodyor antigen. The liposomes are caused to release the enzyme in thepresence of a sample and complement. Antibody- or antigen-labelledliposomes, having water soluble fluorescent or non-fluorescent dyesencapsulated within an aqueous phase or lipid soluble dyes dissolved inthe lipid bilayer of the lipid vesicle or in latex beads, have also beenutilized to assay for analytes capable of entering into animmunochemical reaction with the surface bound antibody or antigen.Detergents have been used to release the dyes from the aqueous phase ofthe liposomes.

[0011] 2. Brief Description of the Related Art

[0012] White, et al. (White), discuss “Chemically Produced ExcitedStates. Energy Transfer, Photochemical Reactions, and Light Emission” inJ. Am. Chem. Soc., 93, 6286 (1971).

[0013] McCapra, et al. (McCapra), disclose “Metal Catalysed LightEmission from a Dioxetan” in Tetrahedron Letters, 23:49, 5225-5228(1982).

[0014] Wildes, et al. (Wildes), discuss “The Dioxetane-SensitizedChemiluminescence of Lanthanide Chelates. A Chemical Source of‘Monochromatic’ Light” in J. Am. Chem. Soc., 93 (23), 6286-6288 (1971).

[0015] Handley, et al. (Handley), disclose “Effects of HeteroatomSubstituents on the Properties of 1,2-Dioxetanes” in TetrahedronLetters, 26, 3183 (1985).

[0016] Zaklika, et al. (Zaklika), discuss “Substituent Effects on theDecompositon of 1,2-Dioxetanes” in J. Am. Chem. Soc., 100, 4916 (1978).

[0017] European Patent Application No. 0,345,776 (McCapra) disclosesspecific binding assays that utilize a sensitizer as a label. Thesensitizers include any moiety which, when stimulated by excitation withradiation of one or more wavelengths or other chemical or physicalstimulus (e.g., electron transfer, electrolysis, electroluminescence orenergy transfer) will achieve an excited state which (a) uponinteraction with molecular oxygen will produce singlet molecular oxygen,or (b) upon interaction with a leuco dye will assume a reduced form thatcan be returned to its original unexcited state by interaction withmolecular oxygen resulting in the production of hydrogen peroxide.Either interaction with the excited sensitizer will, with the additionof reagents, produce a detectible signal.

[0018] European Patent Application No. 0,070,685 (Heller, et al. I)describes a homogeneous nucleic acid hybridization diagnostic bynon-radiative energy transfer.

[0019] A light-emitting polynucleotide hybridization diagnostic methodis described in European Patent. Application No. 0,070,687 (Heller, etal. II).

SUMMARY OF THE INVENTION

[0020] One aspect of the present invention is directed to compositionscomprising (a) a metal chelate comprising a metal selected from thegroup consisting of europium, terbium, dysprosium, samarium, osmium andruthenium in at least a hexacoordinated state and (b) a compound havinga structural portion that is a double bond substituted with two arylgroups, an oxygen atom and an atom selected from the group consisting ofoxygen, sulfur and nitrogen. The aryl groups are characterized in thatone is electron donating with respect to the other. Preferably, thecomposition is incorporated in a latex particulate material.

[0021] Another aspect of the present invention is a compound of theformula:

[0022] wherein X′ is S or NR′ wherein R′ is alkyl or aryl and D and D′are independently selected from the group consisting of alkyl and alkylradical.

[0023] Another aspect of the present invention is a compositioncomprising a latex having incorporated therein a compound of theformula:

[0024] wherein X″ is O, S or NR″ wherein R″ is alkyl or aryl, n is 1 to4, and Ar and Ar′ are independently aryl wherein one of Ar or Ar′ iselectron donating with respect to the other and Y is hydrogen or anorganic radical consisting of atoms selected from the group consistingof C, O, N, S, and P and m is 0 to 2.

[0025] Another aspect of the present invention is a compositioncomprising a latex having incorporated therein Compound 1.

[0026] Another aspect of the present invention is a method fordetermining an analyte which comprises (a) providing in combination (1)a medium suspected of containing an analyte, (2) a photosensitizercapable in its excited state of activating oxygen to a singlet state,where the photosensitizer is associated with a specific binding pair(sbp) member, and (3) one of the above-mentioned compositionsincorporated into a latex particulate material having bound thereto ansbp member, (b) treating the combination with light to excite thephotosensitizer, and (c) examining the combination for the amount ofluminescence emitted therefrom. The amount of luminescence is related tothe amount of analyte in the medium.

[0027] Another aspect of the present invention is a kit comprising inpackaged combination: (1) a composition comprising a suspendible latexparticle comprising one of the above-mentioned compounds and (2) aphotosensitizer. The particle has bound thereto a specific binding pair(sbp) member. The photosensitizer is capable in its excited state ofactivating oxygen to its singlet state.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0028] The present invention is directed to chemiluminescentcompositions that upon activation by singlet oxygen exhibitchemiluminescent emission that rapidly decays, generally having a halflife of 0.5 seconds to 30 minutes, preferably 0.5 to 30 seconds, usuallyless than twenty seconds. In addition, the present chemiluminescentcompositions can exhibit high chemiluminescent quantum yield uponactivation by singlet oxygen, generally 0.1 to 0.9, usually 0.1 to 0.6,preferably 0.2 to 0.4. The chemiluminescent light emitted by the metalchelate after activation in the present compositions generally has awave length of about 550 to 700 nm, usually greater than 600 nm. Thechemiluminescent compositions of the present invention are particularlyuseful in luminescent assays. For example, the long wavelength emissionavoids interference from serum absorption in assays on blood or serumsamples. The high quantum yield improves detectibility and the shortlifetime further improves detectibility by causing all the light that isemitted to be delivered in a short pulse rather than over an extendedperiod of time. This can provide higher light intensity at lower quantumyields.

[0029] The quantum yield of chemiluminescence of the presentchemiluminescent compositions, when activated by singlet oxygen, isgenerally about 10 to 100 fold greater, preferably, 10 to 50 foldgreater, than that observed upon irradiation of the components of thecomposition separately. Furthermore, the rate of decay ofchemiluminescence is significantly enhanced with some of the presentcompositions. These properties render the present compositions extremelyuseful in assays for the determination of analytes.

[0030] Before proceeding further with a description of the specificembodiments of the present invention, a number of terms will be definedand described in detail.

[0031] Metal ligand—a compound in which two or more atoms of the samemolecule can coordinate with a metal to form a metal chelate. The metalchelates that form part of the compositions of the present inventioncomprise a metal selected from the group consisting of europium,terbium, dysprosium, samarium, osmium and ruthenium. One of the abovemetals is coordinated with one or more metal ligands, which may be, forexample, 3-(2-thienoly)-1,1,1-trifluoroacetone (TTA),3-benzoyl-1,1,1-trifluoroacetone (BFTA),3-naphthoyl-1,1,1-trifluoroacetone (NPPTA),2,2-dimethyl-4-perfluorobutyoyl-3-butanone (fod), 2,2′-dipyridyl (bpy),phenanthroline (phen), salicylic acid, phenanthroline carboxylic acid,bipyridylcarboxylic acid, aza crown ethers trioctylphosphine oxide, azacryptands, and so forth. Usually, the metal in the metal chelate is atleast hexacoordinated, but may be octacoordinated or more highlycoordinated depending on the metal ligands. The metal chelate will beuncharged, thus the number of acidic groups provided by its ligands willequal the oxidation state of the metal. Usually, the metal ligands willbe relatively hydrophobic so as to impart solubility of the metalchelate in non-polar solvents. Rare earth metals will usually have anoxidation state of three, ruthenium will have an oxidation state of twoand osmium will have an oxidation state of two. Examplary of such metalchelates, by way of illustration and not limitation, is as follows:

[0032] One TTA in 3(a) or 3(b) can be replaced by one of the following:

[0033] wherein DPP (Diphenylphenanthroline) in 3(b) can be replaced byone of the following:

[0034] Two TTA's in 3(a) and 3(b) can be independently replaced bycompounds selected from the following:

[0035] Three TTA's can be independently replaced by compounds selectedfrom the following:

[0036] Many of these metal ligands and metal chelates are known in theart and many are commercially available. In general, metal chelates canbe prepared from metal ligands by combining the metal chloride with thedesired ratio of metal ligand molecules in an organic solvent such as,e.g., acetonitrile and sufficient base, e.g., pyridine, to take up thereleased hydrochloric acid. For example, metal chelates can be preparedby a procedure such as that described by Shinha, A. P., “Fluorescencesand laser action in rare earth chelates,” Spectroscopy InorganicChemistry, Vol 2, (1971), 255-288.

[0037] Aryl group—an organic radical derived from an aromatichydrocarbon by the removal of one atom and containing one or morearomatic rings, usually one to four aromatic rings, which are generallyfive- or six-member rings such as, e.g., phenyl (from benzene), naphthyl(from naphthalene), biphenylenyl, azulenyl, anthryl, phenanthrenyl,pyridyl, indolyl, benzofuranyl, benzothiophenyl, quinolinyl,isoquinolinyl, carbazolyl, acridinyl, imidazolyl, thiazolyl, pyrazinyl,pyrimidinyl, purinyl, pteridinyl, etc.

[0038] Aralkyl—an organic radical having an alkyl group to which isattached an aryl group, e.g., benzyl, phenethyl, 3-phenylpropyl,1-naphthylethyl, etc.

[0039] Electron donating group—a substituent which when bound to amolecule is capable of polarizing the molecule such that the electrondonating group becomes electron poor and positively charged relative toanother portion of the molecule, i.e., has reduced electron density.Such groups may be, by way of illustration and not limitation, amines,ethers, thioethers, phosphines, hydroxy, oxyanions, mercaptans and theiranions, sulfides, etc.

[0040] Alkyl—a monovalent branched or unbranched radical derived from analiphatic hydrocarbon by removal of one H atom; includes both loweralkyl and upper alkyl.

[0041] Alkyl radical—a substituent formed from two or more alkyl groups,which may be independently lower or upper alkyl groups, linked togetherby a functionality such as an ether, including thioether, an amide, anester and the like.

[0042] Lower Alkyl—alkyl containing from 1 to 5 carbon atoms such as,e.g., methyl, ethyl, propyl, butyl, isopropyl, isobutyl, pentyl,isopentyl, etc.

[0043] Upper Alkyl—alkyl containing more than 6 carbon atoms, usually 6to 20 carbon atoms, such as, e.g., hexyl, heptyl, octyl, etc.

[0044] Alkylidene—a divalent organic radical derived from an aliphatichydrocarbon, such as, for example, ethylidene, in which 2 hydrogen atomsare taken from the same carbon atom.

[0045] Substituted—means that a hydrogen atom of a molecule has beenreplaced by another atom, which may be a single atom such as a halogen,etc., or part of a group of atoms forming a functionality such as asubstituent having from 1 to 50 atoms (other than the requisite hydrogenatoms necessary to satisfy the valencies of such atoms), which atoms areindependently selected from the group consisting of carbon, oxygen,nitrogen, sulfur and phosphorus, and which may or may not be bound toone or more metal atoms.

[0046] Analyte—the compound or composition to be detected. The analytecan be comprised of a member of a specific binding pair (sbp) and may bea ligand, which is monovalent (monoepitopic) or polyvalent(polyepitopic), usually antigenic or haptenic, and is a single compoundor plurality of compounds which share at least one common epitopic ordeterminant site. The analyte can be a part of a cell such as bacteriaor a cell bearing a blood group antigen such as A, B, D, etc., or an HLAantigen or a microorganism, e.g., bacterium, fungus, protozoan, orvirus.

[0047] The polyvalent ligand analytes will normally be poly(aminoacids), i.e., polypeptides and proteins, polysaccharides, nucleic acids,and combinations thereof. Such combinations include components ofbacteria, viruses, chromosomes, genes, mitochondria, nuclei, cellmembranes and the like.

[0048] For the most part, the polyepitopic ligand analytes to which thesubject invention can be applied will have a molecular weight of atleast about 5,000, more usually at least about 10,000. In the poly(aminoacid) category, the poly(amino acids) of interest will generally be fromabout 5,000 to 5,000,000 molecular weight, more usually from about20,000 to 1,000,000 molecular weight; among the hormones of interest,the molecular weights will usually range from about 5,000 to 60,000molecular weight.

[0049] A wide variety of proteins may be considered as to the family ofproteins having similar structural features, proteins having particularbiological functions, proteins related to specific microorganisms,particularly disease causing microorganisms, etc. Such proteins include,for example, immunoglobulins, cytokines, enzymes, hormones, cancerantigens, nutritional markers, tissue specific antigens, etc.

[0050] The following are classes of proteins related by structure:protamines, histones, albumins, globulins, scleroproteins,phosphoproteins, mucoproteins, chromoproteins, lipoproteins,nucleoproteins, glycoproteins, T-cell receptors, proteoglycans, HLA,unclassified proteins, e.g., somatotropin, prolactin, insulin, pepsin,proteins found in the human plasma such as blood clotting factors, otherpolymeric materials such as mucopolysaccharides and polysaccharides,microorganisms such as bacteria, viruses and fungi.

[0051] The monoepitopic ligand analytes will generally be from about 100to 2,000 molecular weight, more usually from 125 to 1,000 molecularweight. The analytes include drugs, metabolites, pesticides, pollutants,and the like. Included among drugs of interest are the alkaloids,steroids, steroid mimetic substances, lactams, aminoalkylbenzenes,benzheterocyclics, purines, those derived from marijuana, hormones,vitamins, prostaglandins, tricyclic antidepressants, anti-neoplastics,antibiotics, nucleosides and nucleotides, miscellaneous individual drugswhich include methadone, meprobamate, serotonin, meperidine, lidocaine,procainamide, acetylprocainamide, propranolol, griseofulvin, valproicacid, butyrophenones, antihistamines, chloramphenicol, anticholinergicdrugs, such as atropine, metabolites related to diseased states includespermine, galactose, phenylpyruvic acid, and porphyrin Type 1,aminoglycosides, polyhalogenated biphenyls, phosphate esters,thiophosphates, carbamates, polyhalogenated sulfenamides.

[0052] For receptor analytes, the molecular weights will generally rangefrom 10,000 to 2×10⁸, more usually from 10,000 to 10⁶. Forimmunoglobulins, IgA, IgG, IgE and IgM, the molecular weights willgenerally vary from about 160,000 to about 10⁶. Enzymes will normallyrange from about 10,000 to 1,000,000 in molecular weight. Naturalreceptors vary widely, generally-being at least about 25,000 molecularweight and may be 10⁶ or higher molecular weight, including suchmaterials as avidin, DNA, RNA, thyroxine binding globulin, thyroxinebinding prealbumin, transcortin, etc.

[0053] The term analyte further includes polynucleotide analytes such asthose polynucleotides defined below. These include m-RNA, r-RNA, t-RNA,DNA, DNA-RNA duplexes, etc. The term analyte also includes receptorsthat are polynucleotide binding agents, such-as, for example,restriction enzymes, activators, repressors, nucleases, polymerases,histones, repair enzymes, chemotherapeutic agents, and the like.

[0054] The analyte may be a molecule found directly in a sample such asa body fluid from a host. The sample can be examined directly or may bepretreated to render the analyte more readily detectable. Furthermore,the analyte of interest may be determined by detecting an agentprobative of the analyte of interest such as a specific binding pairmember complementary to the analyte of interest, whose presence will bedetected only when the analyte of interest is present in a sample. Thus,the agent probative of the analyte becomes the analyte that is detectedin an assay. The body fluid can be, for example, urine, blood, plasma,serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears,mucus, and the like.

[0055] Member of a Specific Binding Pair (“sbp members”)—one of twodifferent molecules, having an area on the surface or in a cavity whichspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of the other molecule. Themembers of the specific binding pair are referred to as ligand andreceptor (antiligand). These will usually be members of an immunologicalpair such as antigen-antibody, although other specific binding pairssuch as biotin-avidin, hormones hormone receptors, nucleic acidduplexes, IgG-protein A, polynucleotide pairs such as DNA-DNA, DNA-RNA,and the like are not immunological pairs but are included in theinvention and the definition of sbp member.

[0056] Polynucleotide—a compound or composition which is a polymericnucleotide having in the natural state about 50 to 500,000 or morenucleotides and having in the isolated state about 15 to 50,000 or morenucleotides, usually about 15 to 20,000 nucleotides, more frequently 15to 10,000 nucleotides. The polynucleotide includes nucleic acids fromany source in purified or unpurified form, naturally occurring orsynthetically produced, including DNA (dsDNA and ssDNA) and RNA, usuallyDNA, and may be t-RNA, m-RNA, r-RNA, mitochondrial DNA and RNA,chloroplast DNA and RNA, DNA-RNA hybrids, or mixtures thereof, genes,chromosomes, plasmids, the genomes of biological material such asmicroorganisms, e.g., bacteria, yeasts, viruses, viroids, molds, fungi,plants, animals, humans, and fragments thereof, and the like.

[0057] Ligand—any organic compound for which a receptor naturally existsor can be prepared.

[0058] Ligand Analog—a modified ligand, an organic radical or analyteanalog, usually of a molecular weight greater than 100, which cancompete with the analogous ligand for a receptor, the modificationproviding means to join a ligand analog to another molecule. The ligandanalog will usually differ from the ligand by more than replacement of ahydrogen with a bond which links the ligand analog to a hub or label,but need not. The ligand analog can bind to the receptor in a mannersimilar to the ligand. The analog could be, for example, an antibodydirected against the idiotype of an antibody to the ligand.

[0059] Receptor (“antiligand”)—any compound or composition capable ofrecognizing a particular spatial and polar organization of a molecule,e.g., epitopic or determinant site. Illustrative receptors includenaturally occurring receptors, e.g., thyroxine binding globulin,antibodies, enzymes, Fab fragments, lectins, nucleic acids, protein A,complement component C1q, and the like.

[0060] Specific binding—the specific recognition of one of two differentmolecules for the other compared to substantially less recognition ofother molecules. Generally, the molecules have areas on their surfacesor in cavities giving rise to specific recognition between the twomolecules. Exemplary of specific binding are antibody-antigeninteractions, enzyme-substrate interactions, polynucleotideinteractions, and so forth.

[0061] Non-specific binding—non-covalent binding between molecules thatis relatively independent of specific surface structures. Non-specificbinding may result from several factors includinghydrophobic-interactions between molecules.

[0062] Antibody—an immunoglobulin which specifically binds to and isthereby defined as complementary with a particular spatial and polarorganization of another molecule. The antibody can be monoclonal orpolyclonal and can be prepared by techniques that are well known in theart such as immunization of a host and collection of sera (polyclonal)or by preparing continuous hybrid cell lines and collecting the secretedprotein (monoclonal), or by cloning and expressing nucleotide sequencesor mutagenized versions thereof coding at least for the amino acidsequences required for specific binding of natural antibodies.Antibodies may include a complete immunoglobulin or fragment thereof,which immunoglobulins include the various classes and isotypes, such asIgA, IgD, IgE, IgG1, IgG2a, IgG2b and IgG3, IgM, etc. Fragments thereofmay include Fab, Fv and F(ab′)₂, Fab′, and the like. In addition,aggregates, polymers, and conjugates of immunoglobulins or theirfragments can be used where appropriate so long as binding affinity fora particular molecule is maintained.

[0063] A substituent having from 1 to 50 atoms (other than the requisitehydrogen atoms necessary to satisfy the valencies of such atoms), whichatoms are independently selected from the group consisting of carbon,oxygen, nitrogen, sulfur and phosphorus—an organic radical; the organicradical has 1 to 50 atoms other than the requisite number of hydrogenatoms necessary to satisfy the valencies of the atoms in the radical.Generally, the predominant atom is carbon (C) but may also be oxygen(O), nitrogen (N), sulfur (S), phosphorus (P), wherein the O, N, S, orP, if present, are bound to carbon or one or more of each other or tohydrogen or a metal atom to form various functional groups, such as, forexample, carboxylic acids, alcohols, thiols, carboxamides, carbamates,carboxylic acid esters, sulfonic acids, sulfonic acid esters, phosphoricacids, phosphoric acid esters, ureas, carbamates, phosphoramides,sulfonamides, ethers, sulfides, thioethers, olefins, acetylenes, amines,ketones, aldehydes, nitrites, and the like. Illustrative of such organicradicals or groups, by way of illustration and not limitation, arealkyl, alkylidine, aryl, aralkyl, and alkyl, aryl, and aralkylsubstituted with one or more of the aforementioned functionalities.

[0064] Linking group—the covalent linkage between molecules. The linkinggroup will vary depending upon the nature of the molecules, i.e.,photosensitizer, chemiluminescent compound, sbp member or moleculeassociated with or part of a particle, being linked. Functional groupsthat are normally present or are introduced on a photosensitizer orchemiluminescent compound will be employed for linking these materialsto an sbp member or a particle such as a latex particle.

[0065] For the most part, carbonyl functionalities will find use, bothoxocarbonyl, e.g., aldehyde and non-oxocarbonyl (including nitrogen andsulfur analogs) e.g., carboxy, amidine, amidate, thiocarboxy andthionocarboxy.

[0066] Alternative functionalities of oxo include active halogen, diazo,mercapto, olefin, particularly activated olefin, amino, phosphoro andthe like. A description of linking groups may be found in U.S. Pat. No.3,817,837, which disclosure is incorporated herein by reference.

[0067] Common functionalities in forming a covalent bond between thelinking group and the molecule to be conjugated are alkylamine, amidine,thioamide, ether, urea, thiourea, guanidine, azo, thioether andcarboxylate, sulfonate, and phosphate esters, amides and thioesters.

[0068] For the most part, the photosensitizer and chemilumenescentcompound will have a non-oxocarbonyl group including nitrogen and sulfuranalogs, a phosphate group, an amino group, alkylating agent such ashalo or tosylalkyl, oxy (hydroxyl or the sulfur analog, mercapto)oxocarbonyl (e.g., aldehyde or ketone), or active olefin such as a vinylsulfone or α,β-unsaturated ester. These functionalities will be linkedto amine groups, carboxyl groups, active olefins, alkylating agents,e.g., bromoacetyl. Where an amine and carboxylic acid or its nitrogenderivative or phophoric acid are linked, amides, amidines andphosphoramides will be formed. Where mercaptan and activated olefin arelinked, thioethers will be formed. Where a mercaptan and an alkylatingagent are linked, thioethers will be formed. Where aldehyde and an amineare linked under reducing conditions, an alkylamine will be formed.Where a carboxylic acid or phosphate acid and an alcohol are linked,esters will be formed.

[0069] Photosensitizer—a sensitizer for generation of singlet oxygenusually by excitation with light. The photosensitizer can bephotoactivatable (e.g., dyes and aromatic compounds) or chemiactivated(e.g., enzymes and metal salts). When excited by light thephotosensitizer is usually a compound comprised of covalently bondedatoms, usually with multiple conjugated double or triple bonds. Thecompound should absorb light in the wavelength range of 200-1100 nm,usually 300-1000 nm, preferably 450-950 nm, with an extinctioncoefficient at its absorbance maximum greater than 500 M⁻¹ cm⁻¹,preferably at least 5000 M⁻¹cm⁻¹, more preferably at least 50,000M⁻¹cm⁻¹ at the excitation wavelength. The lifetime of an excited stateproduced following absorption of light in the absence of oxygen willusually be at least 100 nsec, preferably at least 1 msec. In general,the lifetime must be sufficiently long to permit energy transfer tooxygen, which will normally be present at concentrations in the range of10⁻⁵ to 10⁻³M depending on the medium. The sensitizer excited state willusually have a different spin quantum number (S) than its ground stateand will usually be a triplet (S=1) when, as is usually the case, theground state is a singlet (S═O). Preferably, the sensitizer will have ahigh intersystem crossing yield. That is, photoexcitation of asensitizer will produce the long lived state (usually triplet) with anefficiency of at least 10%, desirably at least 40%, preferably greaterthan 80%. The photosensitizer will usually be at most weakly fluorescentunder the assay conditions (quantum yield usually less that 0.5,preferably less that 0.1).

[0070] Photosensitizers that are to be excited by light will berelatively photostable and will not react efficiently with singletoxygen. Several structural features are present in most usefulsensitizers. Most sensitizers have at least one and frequently three ormore conjugated double or triple bonds held in a rigid, frequentlyaromatic structure. They will frequently contain at least one group thataccelerates intersystem crossing such as a carbonyl or imine group or aheavy atom selected from rows 3-6 of the periodic table, especiallyiodine or bromine, or they may have extended aromatic structures.Typical sensitizers include acetone, benzophenone, 9-thioxanthone,eosin, 9,10-dibromoanthracene, methylene blue, metallo-porphyrins, suchas hematoporphyrin, phthalocyanines, chlorophylls, rose bengal,buckminsterfullerene, etc., and derivatives of these compounds havingsubstituents of 1 to 50 atoms for rendering such compounds morelipophilic or more hydrophilic and/or as attaching groups forattachment, for example, to an sbp member. Examples of otherphotosensitizers that may be utilized in the present invention are thosethat have the above properties and are enumerated in N. J. Turro,“Molecular Photochemistry”, page 132, W.A. Benjamin Inc., N.Y. 1965.

[0071] The photosensitizers are preferably relatively non-polar toassure dissolution into a lipophilic member when the photosensitizer isincorporated in an oil droplet, liposome, latex particle, etc.

[0072] The photosensitizers useful in this invention are also intendedto include other substances and compositions that can produce singletoxygen with or, less preferably, without activation by an external lightsource. Thus, for example, molybdate (MoO₄ ⁼) salts and chloroperoxidaseand myeloperoxidase plus bromide or chloride ion (Kanofsky, J. Biol.Chem. (1983) 259 5596) have been shown to catalyze the conversion ofhydrogen peroxide to singlet oxygen and water. Either of thesecompositions can, for example, be included in particles to which isbound an sbp member and used in the assay method wherein hydrogenperoxide is included as an ancillary reagent, chloroperoxidase is boundto a surface and molybdate is incorporated in the aqueous phase of aliposome. Also included within the scope of the invention asphotosensitizers are compounds that are not true sensitizers but whichon excitation by heat, light, or chemical activation will release amolecule of singlet oxygen. The best known members of this class ofcompounds includes the endoperoxides such as1,4-biscarboxyethyl-1,4-naphthalene endoperoxide,9,10-diphenylanthracene-9,10-endoperoxide and 5,6,11,12-tetraphenylnaphthalene 5,12-endoperoxide. Heating or direct absorption of light bythese compounds releases singlet oxygen.

[0073] Support or Surface—a surface comprised of a porous or non-porouswater insoluble material. The surface can have any one of a number ofshapes, such as strip, rod, particle, including bead, and the like. Thesurface can be hydrophilic or capable of being rendered hydrophilic andincludes inorganic powders such as silica, magnesium sulfate, andalumina; natural polymeric materials, particularly cellulosic materialsand materials derived from cellulose, such as fiber containing papers,e.g., filter paper, chromatographic paper, etc.; synthetic or modifiednaturally occurring polymers, such as nitrocellulose, cellulose acetate,poly (vinyl chloride), polyacrylamide, cross linked dextran, agarose,polyacrylate, polyethylene, polypropylene, poly(4-methylbutene),polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon,poly(vinyl-butyrate), etc.; either used by themselves or in conjunctionwith other materials; glass available as Bioglass, ceramics, metals, andthe like. Natural or synthetic assemblies such as liposomes,phospholipid vesicles, and cells can also be employed.

[0074] Binding of sbp members to the support or surface may beaccomplished by well-known techniques, commonly available in theliterature. See, for example, “Immobilized Enzymes,” Ichiro Chibata,Halsted Press, New York (1978) and Cuatrecasas, J. Biol. Chem., 245:3059(1970).

[0075] Particles—particles of at least about 20 nm and not more thanabout 20 microns, usually at least about 40 nm and less than about 10microns, preferably from about 0.10 to 2.0 microns diameter, normallyhaving a volume of less than 1 picoliter. The particle may be organic orinorganic, swellable or non-swellable, porous or non-porous, having anydensity, but preferably of a density approximating water, generally fromabout 0.7 to about 1.5 g/ml, preferably suspendible in water, andcomposed of material that can be transparent, partially transparent, oropaque. The particles may or may not have a charge, and when they arecharged, they are preferably negative. The particles may be solid (e.g.,polymer, metal, glass, organic and inorganic such as minerals, salts anddiatoms), oil droplets (e.g., hydrocarbon, fluorocarbon, silicon fluid),or vesicles (e.g., synthetic such as phospholipid or natural such ascells and organelles). The particles may be latex particles or otherparticles comprised of organic or inorganic polymers; lipid bilayers,e.g., liposomes, phospholipid vesicles; oil droplets; silicon particles;metal sols; cells; and dye crystallites.

[0076] The organic particles will normally be polymers, either additionor condensation polymers, which are readily dispersible in the assaymedium. The organic particles will also be adsorptive orfunctionalizable so as to bind at their surface, either directly orindirectly, an sbp member and to bind at their surface or incorporatewithin their volume a photosensitizer or a chemiluminescent compound.

[0077] The particles can be derived from naturally occurring materials,naturally occurring materials which are synthetically modified andsynthetic materials. Natural or synthetic assemblies such as lipidbilayers, e.g., liposomes and non-phospholipid vesicles, are preferred.Among organic polymers of particular interest are polysaccharides,particularly cross-linked polysaccharides, such as agarose, which isavailable as Sepharose, dextran, available as Sephadex and Sephacryl,cellulose, starch, and the like; addition polymers, such as polystyrene,polyacrylamide, homopolymers and copolymers of derivatives of acrylateand methacrylate, particularly esters and amides having free hydroxylfunctionalities including hydrogels, and the like. Inorganic polymersinclude silicones, glasses, available as Bioglas, and the like. Solsinclude gold, selenium, and other metals. Particles may also bedispersed water insoluble dyes such as porphyrins, phthalocyanines,etc., which may also act as photosensitizers. Particles may also includediatoms, cells, viral particles, magnetosomes, cell nuclei and the like.

[0078] Where the particles are commercially available, the particle sizemay be varied by breaking larger particles into smaller particles bymechanical means, such as grinding, sonication, agitation, etc.

[0079] The particles will usually be polyfunctional or be capable ofbeing polyfunctionalized or be capable of being bound to an sbp member,photosensitizer, or chemiluminescent compound through specific ornon-specific covalent or non-covalent interactions. A wide variety offunctional groups are available or can be incorporated. Exemplaryfunctional groups include carboxylic acids, aldehydes, amino groups.,cyano groups, ethylene groups, hydroxyl groups, mercapto groups and thelike. When covalent attachment of a sbp member, chemiluminescentcompound or photosensitizer to the particle is employed, the manner oflinking is well known and is amply illustrated in the literature. Seefor example Cautrecasas, J. Biol. Chem., 245:3059 (1970). The length ofa linking group may vary widely, depending upon the nature of thecompound being linked, the nature of the particle, the effect of thedistance between the compound being linked and the particle on thebinding of sbp members and the analyte and the like.

[0080] The photosensitizer can be chosen to dissolve in or noncovalentlybind to the surface of the particles. In this case these compounds willpreferably be hydrophobic to reduce their ability to dissociate from theparticle and thereby cause both compounds to associate with the sameparticle.

[0081] The number of photosensitizer or chemiluminescent moleculesassociated with each particle will on the average usually be at leastone and may be sufficiently high that the particle consists entirely ofphotosensitizer or chemiluminescent molecules. The preferred number ofmolecules will be selected empirically to provide the highest signal tobackground in the assay. In some cases this will be best achieved byassociating a multiplicity of different photosensitizer molecules toparticles. Usually, the photosensitizer or chemiluminescent compound tosbp member ratio in the particles should be at least 1, preferably atleast 100 to 1, and most preferably over 1,000 to 1.

[0082] Latex particles—“Latex” signifies a particulate water suspendiblewater insoluble polymeric material usually having particle dimensions of20 nm to 20 mm, more preferably 100 to 1000 nm in diameter. The latex isfrequently a substituted polyethylene such as polystyrene-butadiene,polyacrylamide polystyrene, polystyrene with amino groups, poly-acrylicacid, polymethacrylic acid, acrylonitrile-butadiene, styrene copolymers,polyvinyl acetate-acrylate, polyvinyl pyrridine, vinyl-chloride acrylatecopolymers, and the like. Non-crosslinked polymers of styrene andcarboxylated styrene or styrene functionalized with other active groupssuch as amino, hydroxyl, halo and the like are preferred. Frequently,copolymers of substituted styrenes with dienes such as butadiene will beused.

[0083] The association of the photosensitizer or chemiluminescentcompound with latex particles utilized in the present invention mayinvolve incorporation during formation of the particles bypolymerization but will usually involve incorporation into preformedparticles, usually by noncovalent dissolution into the particles.Usually a solution of the chemiluminescent compound or sensitizer willbe employed. Solvents that may be utilized include alcohols, includingethanol, ethylene glycol and benzyl alcohol; amides such as dimethylformamide, formamide, acetamide and tetramethyl urea and the like;sulfoxides such as dimethyl sulfoxide and sulfolane; and ethers such ascarbitol, ethyl carbitol, dimethoxy ethane and the like, and water. Theuse of solvents having high boiling points in which the particles areinsoluble permits the use of elevated temperatures to facilitatedissolution of the compounds into the particles and are particularlysuitable. The solvents may be used singly or in combination.Particularly preferred solvents for incorporating photosensitizer arethose that will not quench the triplet excited state of thephotosensitizer either because of their intrinsic properties or becausethey can subsequently be removed from the particles by virtue of theirability to be dissolved in a solvent such as water that is insoluble inthe particles. Aromatic solvents are preferred, and generally solventsthat are soluble in the particle. For incorporating chemiluminescentcompounds in particles a solvent should be selected that does notinterfere with the luminescence because of their intrinsic properties orability to be removed from the particles. Frequently, aromatic solventswill also be preferred. Typical aromatic solvents includedibutylphthalate, benzonitrile, naphthonitrile, dioctylterephthalate,dichlorobenzene, diphenylether, dimethoxybenzene, etc.

[0084] Except when the photosensitizer or chemiluminescent compound isto be covalently bound to the particles, it will usually be preferableto use electronically neutral photosensitizers or chemiluminescentcompounds. It is preferable that the liquid medium selected does notsoften the polymer beads to the point of stickiness. A preferredtechnique comprises suspending the selected latex particles in a liquidmedium in which the photosensitizer or chemiluminescent compound has atleast limited solubility. Preferably, the concentrations of thephotosensitizer and chemiluminescent compound in the liquid media willbe selected to provide particles that have the highest efficiency ofsinglet oxygen formation and highest quantum yield of emission from thechemiluminescent compound in the media but less concentrated solutionswill sometimes be preferred. Distortion or dissolution of the particlesin the solvent can be prevented by adding a miscible cosolvent in whichthe particles are insoluble.

[0085] Generally, the temperature employed during the procedure will bechosen to maximize the singlet oxygen formation ability of thephotosensitizer labeled particles and the quantum yield of thechemiluminescent compound particles with the proviso that the particlesshould not melt or become aggregated at the selected temperature.Elevated temperatures are normally employed. The temperatures for theprocedure will generally range from 20° C. to 200° C., more usually from50° C. to 170° C. It has been observed that some compounds that arenearly insoluble at room temperature, are soluble in, for example, lowmolecular weight alcohols, such as ethanol and ethylene glycol and thelike, at elevated temperatures. Carboxylated modified latex particleshave been shown to tolerate low molecular weight alcohols at suchtemperatures.

[0086] An sbp member may be physically adsorbed on the surface of thelatex particle or may be covalently bonded to the particle. In caseswherein the sbp member is only weakly bound to the surface of the latexparticle, the binding may in certain cases be unable to endureparticle-to-particle shear forces encountered during incubation andwashings. Therefore, it may be preferable to covalently bond sbp membersto the latex particles under conditions that will minimize adsorption.This may be accomplished by chemically activating the surface of thelatex. For example, the N-hydroxysuccinimide ester of surface carboxylgroups can be formed and the activated particles to reduce nonspecificbinding of assay components to the particle surface, are then contactedwith a linker having amino groups that will react with the ester groupsor directly with an sbp member that has an amino group. The linker willusually be selected to reduce nonspecific binding of assay components tothe particle surface and will preferably provide suitable functionalityfor both attachment to the latex particle and attachment of the sbpmember. Suitable materials include maleimidated aminodextran (MAD),polylysine, aminosaccharides, and the like. MAD can be prepared asdescribed by Hubert, et al., Proc. Natl. Acad. Sci., 75(7), 3143, 1978.

[0087] In one method, MAD is first attached to carboxyl-containing latexparticles using a water soluble carbodiimide, for example,1-(3-dimethylaminopropyl)-3-ethyl carbodiimide. The coated particles arethen equilibrated in reagents to prevent nonspecific binding. Suchreagents include proteins such as bovine gamma globulin (BGG), anddetergent, such as Tween 20, TRITON X-100 and the like. A sbp memberhaving a sulfhydryl group, or suitably modified to introduce asulfhydryl group, is then added to a suspension of the particles,whereupon a covalent bond is formed between the sbp member and the MADon the particles. Any excess unreacted sbp member can then be removed bywashing.

[0088] Chemiluminescent compound—compounds that form part of thecompositions of the present invention are enol ethers generally havingthe structural portion selected from the group consisting of:

[0089] wherein Ar and Ar′ are independently aryl wherein one of Ar orAr′, preferably Ar, is electron donating with respect to the other. Thismay be achieved, for example, by the presence of one or more electrondonating groups in one of Ar or Ar′. The part of the above structuresrepresented by the broken lines are not critical to the presentinvention and may be any substituent as long as such substituent doesnot interfere with dioxetane formation and transfer of energy.Generally, the compounds are those of Compound 2 wherein, preferably, mis 0, and n is 1 to 3.

[0090] For the most part the compounds that form part of the presentcomposition have the structural portion:

[0091] wherein X is O, S or N wherein the valency of N is completed withhydrogen or an organic radical consisting of atoms selected from thegroup consisting of C, O, N, S, and P and Ar and Ar′ are independentlyaryl wherein one of Ar or Ar′ is electron donating with respect to theother.

[0092] The broken lines in the above structure signify that the ring canbe independently unsubstituted or substituted with a substituent havingfrom 1 to 50 atoms. In addition, the substituents may be taken togetherto form a ring such as, for example, aryl, which may in turn besubstituted with a substituent having from 1 to 50 atoms.

[0093] Exemplary enol ethers, by way of illustration and not limitation,are set forth in the following chart with reference to the followingstructure:

[0094] wherein Compounds 9-17 have the following moieties for X, Ar, andAr′. X Ar Ar′ * O

9 S

10 S

11 S

12 S

13 S

14 S

15

16

17

[0095] The chemiluminescent compounds undergo a chemical reaction withsinglet oxygen to form a metastable intermediate that can decompose withthe simultaneous or subsequent emission of light within the wavelengthrange of 250 to 1200 nm. Preferably, the intermediate decomposesspontaneously without heating or addition of ancillary reagentsfollowing its formation. However, addition of a reagent after formationof the intermediate or the use of elevated temperature to acceleratedecomposition will be required for some chemiluminescent compounds. Thechemiluminescent compounds are usually electron rich compounds thatreact with singlet oxygen, frequently with formation of dioxetanes ordioxetanones, such as those represented by the following structure wherethe substituents on the carbon (C) atoms are those present on thecorresponding olefin:

[0096] some of which decompose spontaneously, others by heating and/orby catalysis usually by an electron rich energy acceptor, with theemission of light. For some cases the dioxetane is spontaneouslyconverted to a hydroperoxide whereupon basic pH is required to reformthe dioxetane and permit decomposition and light emission.

[0097] The chemiluminescent compounds of interest will generally emit atwavelengths above 300 nanometers and usually above 400 nm. Compoundsthat alone or together with a fluorescent molecule emit light atwavelengths beyond the region where serum components absorb light willbe of particular use in the present invention. The fluorescence of serumdrops off rapidly above 500 nm and becomes relatively unimportant above550 nm. Therefore, when the analyte is in serum, chemiluminescentcompounds that emit light above 550 nm, preferably above 600 nm are ofparticular interest. In order to avoid autosensitization of thechemiluminescent compound, it is preferable that the chemiluminescentcompounds do not absorb light used to excite the photosensitizer. Sinceit will generally be preferable to excite the sensitizer with lightwavelengths longer than 500 nm, it will therefore be desirable thatlight absorption by the chemiluminescent compound be very low above 500nm.

[0098] The chemiluminescent compounds of the present invention can beprepared in a number of different ways. In one approach a 2-thioethanolderivative is condensed with an appropriate diaryl substitutedalpha-hydroxy ketone (substituted benzoin) where one aryl is substitutedon the ketone carbon and the other is substituted on the carboncontaining the alpha-hydroxy group. The condensation reaction yields theappropriate enol ether directly. The above condensation can be carriedout in an inert solvent such as toluene. Usually, the temperature of thereaction is about 90-130° and the reaction is allowed to proceed for aperiod of 5-50 hours. Generally, the reaction is carried out at thereflux temperature of the combined reagents. The condensation is carriedout in the presence of a Lewis acid, for example, an acyl chloride,silyl chloride, stannous chloride, etc. The following reaction scheme isillustrative of the above-described method for preparing thechemiluminescent compounds of the present invention:

[0099] Another reaction scheme for preparing compounds in accordancewith the present invention, particularly those containing an alkylradical, is depicted in the following schematic for synthesizingCompound 13:

Synthesis of C-26 Thioxene (Compound 13)

[0100]

[0101] In the above synthesis ethyl 5-bromovalerate is condensed withN-methylaniline to give 22 which is converted by Kilsmeier-Haaksynthesis (DMF/POCl₃) to aldehyde 23. Benzoin condensation of 23 withbenzaldehyde yields 24 which is hydrolyzed with potassium hydroxide andconverted to amide 25 with didecylamine and diphenylphosphoryl azide(DPPA). Conversion to Compound 13 was carried out by condensation withmercaptoethanol and trimethylsilylchloride.

[0102] Another approach for preparing compounds in accordance with thepresent invention, particularly involving regioselective synthesis isshown in the following schematic for synthesizing Compound 14:

[0103] In the above synthesis reaction of p-nitrophenylacetic acid (27)with decanal in the presence of pd/carbon and hydrogen gas at 100 psigives didecylamine 28, which is condensed with p-heptylbenzene to giveketone 29. Bromine and trifluoroacetic acid are used to brominate 29 andbicarbonate converts the product to benzoin 30. Conversion to Compound14 is carried out by condensation with mercaptoethanol andtrimethylsilylchloride.

[0104] Ancillary Materials—Various ancillary materials will frequentlybe employed in the assay in accordance with the present invention. Forexample, buffers will normally be present in the assay medium, as wellas stabilizers for the assay medium and the assay components.Frequently, in addition to these additives, proteins may be included,such as albumins, organic solvents such as formamide, quaternaryammonium salts, polycations such as dextran sulfate, surfactants,particularly non-ionic surfactants, binding enhancers, e.g.,polyalkylene glycols, or the like. When the photosensitizer is activatedchemically rather than by irradiation, hydrogen peroxide will often beincluded as an ancillary reagent. When it is desired to shift theemission wavelength of the chemiluminescent compound to longerwavelength or catalyse the decomposition of its oxygen-activated form, afluorescent molecule may be employed.

[0105] Wholly or Partially Sequentially—when the sample and variousagents utilized in the present invention are combined other thanconcomitantly (simultaneously), one or more may be combined with one ormore of the remaining agents to form a subcombination. Eachsubcombination can then be subjected to one or more steps of the presentmethod. Thus, each of the subcombinations can be incubated underconditions to achieve one or more of the desired results.

[0106] One aspect of the present invention is directed to compositionscomprising (a) a metal chelate comprising a metal selected from thegroup consisting of europium, terbium, dysprosium, samarium, osmium andruthenium in at least a hexacoordinated state and (b) a compound havinga structural portion that is a double bond substituted with two arylgroups, an oxygen atom and an atom selected from the group consisting ofoxygen, sulfur and nitrogen. The aryl groups are characterized in thatone is electron donating with respect to the other. The composition ofthe present invention comprising a metal chelate and an olefiniccompound is generally in a medium that may be liquid or solid, usuallysolid particulate. The liquid medium is usually a high-boiling, waterimmisinble liquid such as one from the group comprising toluene, lipids,fluorocarbons, diphenylether, chlorobenzene, dioctylphthalate,dimethoxybenzene, mineral oil and triacylglycerides and the solidparticulate medium can be an organic polymer such as polystyrene,polymethylacrylate, polyacrylate, polyacrylamide, polyvinylchloride andcopolymers thereof, nylon and other polyamides, etc. Preferably, thecomposition is incorporated in a latex particulate material.

[0107] The metal chelate is present in an amount to maximize thechemiluminescent quantum yield and minimize the decay time ofchemiluminescence. Usually, the metal chelate is present at 0.2-500 mM,preferably 2-100 mM. In some circumstances, usually when the metalchelate is hexacoordinated, reduction in the decay time is accompaniedby a reduction in quantum yield and a balance must be reached betweenthese two effects. Accordingly, the concentration of the metal chelatein the composition should be adjusted to achieve such a balance. Theconcentration of the chemiluminescent compound in the composition isusually 0.1-500 mM, preferably 2-100 mM.

[0108] Preferred compounds of the present invention have the formula ofCompound 1. Representative of such compounds are Compounds 10-16.Particularly preferred compounds are those of the formula of Compound 1wherein X′ is S or NR′ wherein R′ is lower alkyl or aryl and D and D′are independently lower alkyl, preferably wherein X′ is S. Particularlypreferred compounds within the above are those wherein D and D′ aremethyl and R′ is methyl or phenyl, and a most preferred compound is onein which X′ is S and D and D′ are methyl. Compound 13 is one of the morepreferred of the above compounds.

[0109] One aspect of the present invention is a composition comprising alatex having incorporated therein Compound 2. Preferred compositions arethose wherein R′ is methyl or phenyl and wherein n is 1 or 2 and m is 0.Preferably, Ar is selected from the group consisting of 5-member and6-member aromatic and heteroaromatic rings. In a preferred embodiment Aris phenyl substituted with an electron donating group at a position ofthe phenyl that is meta or para to the carbon that is bonded to thedouble bond and Ar′ is phenyl. Exemplary compositions are thosecontaining a compound selected from the group consisting of Compounds9-16. The latex particles are usually suspendible and have an averagediameter of 0.04 to 4000 nanometer. For assays the particle will have anspb member bound to it and will have an average diameter of 100 to 1000micrometers.

[0110] Another embodiment of the present invention is a method fordetermining an analyte. The method comprises (a) providing incombination (1) a medium suspected of containing an analyte, (2) aphotosensitizer capable in its excited state of activating oxygen to asinglet state, the photosensitizer associated with a specific bindingpair (sbp) member, and (3) a suspendible latex particulate materialcomprising Compound 2. The particulate material has bound thereto an sbpmember. The combination is treated with light, usually by irradiation,to excite the photosensitizer, and is then examined for the amount ofluminescence emitted. The amount of such luminescence is related to theamount of analyte in the medium. The photosensitizer may be incorporatedin a second suspendible particulate material. Particularly usefulcompositions for determining an analyte in accordance with the presentinvention are those containing Compound 1.

[0111] In the assay protocol the components are provided in combinationand the light produced as a function of activation of oxygen by thesensitizer will be a function of analyte concentration. Advantageously,the methods of the present invention can be carried out without heatingthe medium to produce light. Consequently, the assay of the presentinvention can be conducted at a constant temperature.

[0112] The chemiluminescent compound may be bound to a sbp member thatis capable of binding directly or indirectly to the analyte or to anassay component whose concentration is affected by the presence of theanalyte. The term “capable of binding directly or indirectly” means thatthe designated entity can bind specifically to the entity (directly) orcan bind specifically to a specific binding pair member or to a complexof two or more sbp members which is capable of binding the other entity(indirectly). Preferably, assays conducted in accordance with thepresent invention utilize one of the above compositions in a latexparticle. This latex particle has an sbp member generally capable ofbinding directly or indirectly to the analyte or a receptor for theanalyte. When the sbp members associated with the photosensitizer andthe chemiluminescent compound are both capable of binding to theanalyte, a sandwich assay protocol results. When one of the sbp membersassociated with the photosensitizer or chemiluminescent compound canbind both the analyte and an analyte analog, a competitive assayprotocol can result.

[0113] The photosensitizer is usually caused to activate thechemiluminescent compound by irradiating the medium containing the abovereactants. The medium must be irradiated with light having a wavelengthwith energy sufficient to convert the photosensitizer to an excitedstate and thereby render it capable of activating molecular oxygen tosinglet oxygen. The excited state for the photosensitizer capable ofexciting molecular oxygen is generally a triplet state which is morethan about 20, usually at least 23, Kcal/mol more energetic than thephotosensitizer ground state. Preferably, the medium is irradiated withlight having a wavelength of about 450 to 950 nm although shorterwavelengths can be used, for example, 230-950 nm. The luminescenceproduced may be measured in any convenient manner such asphotographically, visually or photometrically to determine the amountthereof, which is related to the amount of analyte in the medium.

[0114] Although it will usually be preferable to excite thephotosensitizer by irradiation with light of a wavelength that isefficiently absorbed by the photosensitizer, other means of excitationmay be used as for example by energy transfer from an excited state ofan energy donor such as a second photosensitizer. When a secondphotosensitizer is used, wavelengths of light can be used which areinefficiently absorbed by the photosensitizer but efficiently absorbedby the second photosensitizer. The second photosensitizer may be boundto an assay component that is associated, or becomes associated, withthe first photosensitizer, for example, bound to a surface orincorporated in the particle having the first photosensitizer. When asecond photosensitizer is employed it will usually have a lowest energysinglet state at a higher energy than the lowest energy singlet state ofthe first photosensitizer.

[0115] The 632.6 nm emission line of a helium-neon laser is aninexpensive light source for excitation. Photosensitizers withabsorption maxima in the region of about 620 to about 650 nm arecompatible with the emission line of a helium-neon laser and are,therefore, particularly useful in the present invention.

[0116] The method and compositions of the invention may be adapted tomost assays involving sbp members such as ligand-receptor; e.g.,antigen-antibody reactions; polynucleotide binding assays, and so forth.The assays may be homogeneous or heterogeneous, competitive ornoncompetitive. The assay components, chemiluminescent compound andphotosensitizer, can be utilized in a number of ways with (1) a surface,when employed, (2) nucleic acid or receptor and (3) nucleic acid orligand. The association may involve covalent or non-covalent bonds.Those skilled in the art will be able to choose appropriate associationsdepending on the particular assay desired in view of the foregoing andthe following illustrative discussion.

[0117] In a homogeneous assay approach, the sample may be pretreated ifnecessary to remove unwanted materials. The reaction for anoncompetitive sandwich type assay can involve an sbp member, (e.g., anantibody, nucleic acid probe, receptor or ligand) complementary to theanalyte and associated with a chemiluminescent compound; aphotosensitizer associated with an sbp member, (e.g., antibody, nucleicacid probe, receptor or ligand) that is also complementary to theanalyte; the sample of interest; and any ancillary reagents required.Preferably, at least the chemiluminescent compound is incorporated inparticles to which an sbp member is attached. The photosensitizer may bedirectly attached to an sbp member or it may also be incorporated intoparticles. In a competitive protocol one sbp member can be a derivativeof the analyte and the other sbp member can be complementary to theanalyte, e.g., an antibody. In either protocol the components may becombined either simultaneously or wholly or partially sequentially. Theability of singlet oxygen produced by an activated photosensitizer toreact with the chemiluminescent compound is governed by the binding ofan sbp member to the analyte. Hence, the presence or amount of analytecan be determined by measuring the amount of light emitted uponactivation of the photosensitizer by irradiation, heating or addition ofa chemical reagent, preferably by irradiation. Both the binding reactionand detection of the extent thereof can be carried out in a homogeneoussolution without separation. This is an advantage of the presentinvention over prior art methods utilizing chemiluminescence.

[0118] In a heterogeneous assay approach, the assay components comprisea sample suspected of containing an analyte which is an sbp member; ansbp member bound to a support, which may be either a non-dispersiblesurface or a particle having associated with it one member of a groupconsisting of the chemiluminescent compound and the photosensitizer; andan sbp member having the other member of the group associated with itwherein the sbp members can independently, either directly orindirectly, bind the analyte or a receptor for the analyte. Thesecomponents are generally combined either simultaneously or wholly orpartially sequentially. The surface or particles are then separated fromthe liquid phase and either the separated phase or the liquid phase issubjected to conditions for activating the photosensitizer, usually byirradiating the particular phase in question, and measuring the amountof light emitted.

[0119] The binding reactions in an assay for the analyte will normallybe carried out in an aqueous medium at a moderate pH, generally thatwhich provides optimum assay sensitivity. Preferably, the activation ofthe photosensitizer will also be carried out in an aqueous medium.However, when a separation step is employed, non-aqueous media such as,e.g., acetonitrile, acetone, toluene, benzonitrile, etc. and aqueousmedia with pH values that are very high, i.e., greater than 10.0, orvery low, i.e., less than 4.0, usually very high, can be used. Asexplained above, the assay can be performed either without separation(homogeneous) or with separation (heterogeneous) of any of the assaycomponents or products.

[0120] The aqueous medium may be solely water or may include from 0.01to 80 volume percent of a cosolvent but will usually include less than40% of a cosolvent when an sbp member is used that is a protein. The pHfor the medium of the binding reaction will usually be in the range ofabout 4 to 11, more usually in the range of about 5 to 10, andpreferably in the range of about 6.5 to 9.5. When the pH is not changedduring the generation of singlet oxygen the pH will usually be acompromise between optimum binding of the binding members and the pHoptimum for the production of signal and the stability of other reagentsof the assay. When elevated pH's are required for signal production, astep involving the addition of alkaline reagent can be inserted betweenthe binding reaction and generation of singlet oxygen and/or signalproduction. Usually the elevated pH will be greater than 10, usually10-14. For heterogenous assays non-aqueous solvents may also be used asmentioned above, the main consideration being that the solvent not reactefficiently with singlet oxygen.

[0121] Various buffers may be used to achieve the desired pH andmaintain the pH during an assay. Illustrative buffers include borate,phosphate, carbonate, tris, barbital and the like. The particular bufferemployed is not critical to this invention, but in an individual assayone or another buffer may be preferred.

[0122] Moderate temperatures are normally employed for carrying out thebinding reactions of proteinaceous ligands and receptors in the assayand usually constant temperature, preferably, 250 to 400, during theperiod of the measurement. Incubation temperatures for the bindingreaction will normally range from about 5° to 45° C., usually from about15° to 40° C., more usually 25° to 40° C. Where binding of nucleic acidsoccur in the assay, higher temperatures will frequently be used, usually20° to 90°, more usually 35° to 75° C. Temperatures during measurements,that is, generation of singlet oxygen and light detection, willgenerally range from about 20° to 100°, more usually from about 25° to50° C., more usually 25° to 40° C.

[0123] The concentration of analyte which may be assayed will generallyvary from about 10⁻⁴ to below 10⁻¹⁶ M, more usually from about 10⁻⁶ to10⁻¹⁴ M. Considerations, such as whether the assay is qualitative,semiquantitative or quantitative, the particular detection technique theconcentration of the analyte of interest, and the maximum desiredincubation times will normally determine the concentrations of thevarious reagents.

[0124] In competitive assays, while the concentrations of the variousreagents in the assay medium will generally be determined by theconcentration range of interest of the analyte, the final concentrationof each of the reagents will normally be determined empirically tooptimize the sensitivity of the assay over the range. That is, avariation in concentration of the analyte which is of significanceshould provide an accurately measurable signal difference.

[0125] The concentration of the sbp members will depend on the analyteconcentration, the desired rate of binding, and the degree that the sbpmembers bind nonspecifically. Usually, the sbp members will be presentin at least the lowest expected analyte concentration, preferably atleast the highest analyte concentration expected, and for noncompetitiveassays the concentrations may be 10 to 10⁶ times the highest analyteconcentration but usually less than 10⁻⁴ M, preferably less than 10⁻⁶ M,frequently between 10⁻¹¹ and 10⁻⁷ M. The amount of photosensitizer orchemiluminescent compound associated with a sbp member will usually beatleast one molecule per sbp member and may be as high as 10⁵, usually atleast 10-10⁻⁴ when the photosensitizer or chemiluminescent molecule isincorporated in a particle.

[0126] While the order of addition may be varied widely, there will becertain preferences depending on the nature of the assay. The simplestorder of addition is to add all the materials simultaneously.Alternatively, the reagents can be combined wholly or partiallysequentially. When the assay is competitive, it will often be desirableto add the analyte analog after combining the sample and an sbp membercapable of binding the analyte optionally, an incubation step may beinvolved after the reagents are combined, generally ranging from about30 seconds to 6 hours, more usually from about 2 minutes to 1 hourbefore the sensitizer is caused to generate singlet oxygen and the lightemission is measured.

[0127] In a particularly preferred order of addition, a first set ofspecific binding pair members that are complementary to and/orhomologous with the analyte are combined with the analyte followed bythe addition of specific binding pair members complementary to the firstspecific binding pair members, each associated with a different memberof the group consisting of a photosensitizer and a composition of thepresent invention. The assay mixture, or a separated component thereof,is then irradiated and the light emission is measured.

[0128] In a homogeneous assay after all of the reagents have beencombined, they can be incubated, if desired. Then, the combination isirradiated and the resulting light emitted is measured. The emittedlight is related to the amount of the analyte in the sample tested. Theamounts of the reagents of the invention employed in a homogeneous assaydepend on the nature of the analyte. Generally, the homogeneous assay ofthe present invention exhibits an increased sensitivity over knownassays such as the EMIT® assay. This advantage results primarily becauseof the improved signal to noise ratio obtained in the present method.

[0129] Another aspect of the present invention relates to kits usefulfor conveniently performing an assay method of the invention fordetermining the presence or amount of an analyte in a sample suspectedof containing the analyte. The kits comprise in packaged combination:(1) a composition comprising a suspendible latex particle comprising acompound of the formula of Compound 2, preferably of Compound 1, wherethe particle can bind a specific binding pair (sbp) member, and (2) aphotosensitizer capable in its excited state of activating oxygen to itssinglet state. The photosensitizer can be part of a compositioncomprising a second suspendible particle comprising the photosensitizerwhere the second particle has bound thereto a sbp member or it may bedirectly bound to a sbp member. The kit can further include a writtendescription of a method in accordance with the present invention andinstructions for using the reagents of the kit in such method.

[0130] To enhance the versatility of the subject invention, the reagentscan be provided in packaged combination, in the same or separatecontainers, so that the ratio of the reagents provides for substantialoptimization of the method and assay. The reagents may each be inseparate containers or various reagents can be combined in one or morecontainers depending on the cross-reactivity and stability of thereagents. The kit can further include other separately packaged reagentsfor conducting an assay including ancillary reagents, and so forth.

EXAMPLES

[0131] The invention is demonstrated further by the followingillustrative examples. Parts and percentages used herein are by weightunless otherwise specified. Temperatures are in degrees centigrade (°C.).

[0132] Abbreviations:

[0133] Ab_(F) (anti-fluorescein)—Mouse monoclonal antibody tofluorescein.

[0134] Ab_(T3) (anti-T₃)—mouse monoclonal antibody to T₃

[0135] t-Bu—tert-butyl

[0136] TFA—trifluoroacetic acid

[0137] T₃—

[0138] Φ—chemiluminescence quantum yield

[0139] PMT—

[0140] EtoAc—ethyl acetate

[0141] BSA—Bovine serum albumin

[0142] Chl-a—Chlorophyll-a

[0143] D-H₂O—dionized water

[0144] DPP—4,7-Diphenylphenanthroline

[0145] DPPC—dipalmitoylphosphatidyl choline

[0146] DPPG—dipalmitoylphosphatidyl glycerol

[0147] DPPE—dipalmitoylphosphatidyl ethanolamine

[0148] EDAC—1-Ethyl-3-(3-Dimethylaminopropyl) carbodiimidehydrochloride.

[0149] nC₁₀—tetra-(n-decyl)phthalocyanin aluminum chloride complex.

[0150] PB—Polystyrene beads

[0151] PB/nC₁₀—PB containing nC₁₀

[0152] PBS—phosphate buffered saline 0.02M NaPi, 0.14 M NaCl/pH 7.2

[0153] Pi—Phosphate

[0154] Sulfo-NHS—Sulfo-N-hydroxysuccinimide

[0155] SATA —S-acetylthioglycolic acid N-hydroxysuccinimide ester

[0156] RLU—Relative light units.

[0157] NHS—N-hydroxysuccinimide

[0158] DMSO—dimethyl sulfoxide

[0159] DMF—dimethyl formamide

[0160] DCC—dicyclohexylcarbodiimide

[0161] TEA—triethylamine

[0162] TLC—thin layer chromatography

[0163] TNBSA—2,4,6-trinitrobenzenesulfonic acid

[0164] BGG—bovine gamma globulin

[0165] TMSCl—trimethylsilyl chloride

[0166] MeOH—methanol

[0167] Biotin-LC₇—NHS-sulfosuccinimidyl-6-(biotinamido)-hexanoate

[0168] λmax ABS—lambda maximum of absorption

[0169] λmax EMI—lambda maximum of fluorescence emission

[0170] λmax CH.EM.—lambda maximum of chemiluminescence emission

[0171] All monoclonal antibodies were produced by standard hybrid celltechnology. Briefly, the appropriate immunogen was injected into a host,usually a mouse or other suitable animal, and after a suitable period oftime the spleen cells from the host were obtained. Alternatively,unsensitized cells from the host were isolated and directly sensitizedwith the immunogen in vitro. Hybrid cells were formed by fusing theabove cells with an appropriate myeloma cell line and culturing thefused cells. The antibodies produced by the cultured hybrid cells werescreened for their binding affinity to the particular antigen, e.g. TSHor HCG. A number of screening techniques were employed such as, forexample, ELISA screens. Selected fusions were then recloned.

Example 1 Total Triiodothyronine Assay

[0172] I. Bead Preparations

[0173] Materials

[0174] 175 nm Carboxylate modified latex (CML beads) from BangsLaboratories.

[0175] Ethylene glycol, ethoxy ethanol, benzyl alcohol, chlorophyll-afrom Aldrich.

[0176] Europium (III) thienoyl trifluoroacetonate (EuTTA) from Kodak.

[0177] Trioctyl phosphine oxide (TOPO) from Aldrich.

[0178] Dioxene [1-(4-dimethylaminophenyl)-6-phenyl 1,4 dioxene]:Prepared by a modification of a procedure described in: Giagnon, S. D.(1982) University Microfilms International (Ann Arbor, Mich.)

[0179] Procedures

[0180] 1. Chlorophyll-a Sensitizer Beads

[0181] A solution of chlorphyll-a in benzyl alcohol (1.0 mL, 0.6 mM) wasadded to 8.0 mL of benzyl alcohol at 105° C. A suspension of carboxylatemodified latex, 175 nm size, in water (10%, 1.0 mL) was added to thebenzyl alcohol solution. The mixture was stirred for 5 min at 105° C.,and cooled to room temperature. Ethanol (10.0 mL) was added and themixture centrifuged. The pellet was resuspended in a 1:1 ethanol-watermixture (10.0 mL) and the suspension centrifuged. The same resuspensionand centrifugation procedure was repeated in water (10.0 mL), and thepellet was resuspended in water (1.8 mL).

[0182] Characterization

[0183] A. Dye concentration: A solution prepared by adding 10 μL of theabove bead suspension to dioxane (990 μL) was found to have anabsorbance of 0.11 at 660 nm, corresponding to 2.6 μmoles ofChlorophyll-a in one gram of beads.

[0184] B. Singlet oxygen generation: A mixture of chlorphyll-a beads(200 μg) 2×10⁻⁴ moles of anthracene 9,10-dipropionic acid (ADPA) in twomL of phosphate buffer (50 mM, pH 7.5, containing 100 mM NaCl) wasirradiated with a tungsten-halogen lamp equipped with a 645 nm cut-offfilter for 20 min. The beads were removed by filtration, and theconcentration of the oxygenation product was determinedspectrophotometrically at 400 nm. The rate was found to be 3.0 nmoles ofoxygenation product per min. Under the same conditions, 0.38 pmoles of asoluble sensitizer, aluminum phthalocyanin tetrasulfonate generated thesame amount of oxygenation product (the amount of sensitizer in thebeads was 200·10⁻⁶·2.6·10⁻⁶=520 pmoles).

[0185] 2. Chlorophyll-a/Tetrabutyl Squarate Sensitizer Beads

[0186] A suspension of carboxylated latex beads (175 nm size, 10% solidsin water, 30.0 mL) was centrifuged. The supernatant was discarded andthe pellet was resuspended in ethylene glycol (60.0 mL). The suspensionwas heated to 100° C. 9.0 mL of a benzyl alcohol solution which is 1.67mM in Chlorophyll-a and 3.33 mM in tetrabutyl squarate [1,3bis(4-dibutylaminophenyl)square] was added slowly over 3 min to thesuspension. The heating was continued for 7 min, then the suspension wascooled to room temperature in a water bath. The benzyl alcoholsuspension was added to cold ethanol (120 mL). The mixture wascentrifuged and the supernatant discarded. The pellet was resuspended in50% ethanol in water and the suspension was centrifuged. The sameresuspension and centrifugation procedure was repeated in 5 ethanol inwater (30 mL).

[0187] Characterization

[0188] A. Dye concentration. The concentration of the tetrabutylsquarate in the beads was determined spectrophotometrically as describedabove for the chlorophyll-a beads. It was found to be 44 μM dye in thebeads.

[0189] B. Singlet oxygen generation. Twenty-five μL of a 5% mM solutionof ADPA in ethanol were added to suspension of beads (100 μg) inphosphate buffer, pH 7.0 (20 mM, containing 50 mM NaCl). The mixture wasirradiated as above, using a 610 nm long pass filter. The rate ofsinglet oxygen formation was calculated from the rate of the decrease inabsorbance (at 400 nm) of the ADPA. It was found that the beadsgenerated. 7·10⁻² μmoles of singlet oxygen/min.

[0190] 3. Dioxene/EuTTA/TOPO Acceptor Beads

[0191] 20 mL of 175 nm carboxylated latex beads (10% suspension inwater) was added to ethoxy ethanol (20.0 mL). The mixture was heated to90° C. 20 mL of a solution which is 10 mM2-(p-dimethylaminophenyl)-3-phenyl dioxene, 20 mM EuTTA and 60 mM TOPOin ethoxy ethanol were added to the mixture. The heating was continuedfor 7 min at a temperature up to 97° C. The mixture was cooled to roomtemperature. Ethanol (40.0 mL) was added and the mixture wascentrifuged. The pellet was resuspended in 80 ethanol and the suspensionwas centrifuged. The resuspension and centrifugation procedure wasrepeated in 10% ethanol (36 mL).

[0192] Characterization

[0193] A. Dye concentration. The concentration of EuTTA in the beads wasdetermined spectrophotometrically and was found to be 0.07M. Because theconcentration of dioxene cannot be determined in the presence of EuTTA,it was measured in beads which were dyed with the dioxene only,2-(p-dimethylaminophenyl)-3-phenyl dioxene, under the same conditions.The concentration was found to be 0.016M.

[0194] B. Signal generation. A suspension of beads (25 μg) in phosphatebuffer (0.5 mL, 20 mM phosphate, 50 mM NaCl, 0.1% Tween 20, pH 7.0) wasmixed with a solution of 2 μM aluminum phthalocyanine tetrasulfonate(0.5 mL) in the same buffer. The mixture was illuminated for one minutewith a 125 w tungsten-halogen lamp equipped with a 610 nm long passfilter. Following illumination, the mixture was placed in a TurnerTD-20e luminometer, and the luminescence was measured for 20 sec. Theintensity was found to be 327 RLU (relative light unit)/sec. Thewavelength of the emitted light was measured using Perkin-Elmer 650-40scanning spectrofluorimeter. The major emission peak was centered near615 nm.

[0195] II. Assay Procedure

[0196] EDAC/NHS Coupling of Antibody to 40 nm Beads

[0197] 73.6 mg sulfo-NHS(N-hydroxysulfo-succinimide, Pierce Chemical Co.#24510 G) was dissolved in 6 mL of a suspension of 4 mg/mLcarboxylate-modified 40 nm polystyrene beads (dyed with chlorophyl-a andtetrabutyl squarate) in water. 136 uL 0.5 M Na₂HPO₄ was added. PH wasadjusted to 5.2. 136 uL additional water was added. 130.4 mg EDAC(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, SigmaChemical Co. #E-6383) in 454 μL water was slowly added to stirring beadsuspension. The suspension was incubated for 20 min at room temperature.The beads were centrifuged for 20 min. at 15,000 rpm in Sorvall SA-600rotor at 4° C. The supernatant was discarded. The beads were thenresuspended in 1.2 mL 5 mM sodium phosphate, pH 5.8, and the suspensionwas sonicated to redisperse beads. The beads were slowly added to 4.8 mLof a stirring solution containing 1.7 mg/mL IgG (mouse monoclonalanti-fluorescein) and 6.7 mg/mL BSA and 17 mM borax, pH 9.2, and mixedgently overnight at 4° C. 800 uL 2 M glycine was added which was thenfollowed by 2.8 mL 50 mg/mL BSA in 0.1 M borax to the bead suspension.The suspension was sonciated and allowed to mix gently for 3 h at 4° C.The beads were centrifuged for 30 min at 15,000 rpm. The supernatant wasdiscarded. The beads were resuspended in 3 mL 50 mM sodium phosphate and150 mM NaCl, pH 7.6, and the suspension was sonciated. Thecentrifugation, resuspension and sonification steps were repeated for atotal of three spins. After the third spin, beads were resuspended in2.4 mL 50 mM sodium phosphate and 150 mM NaCl, pH 7.6. The resultingsuspension was sonicated and stored at 4° C.

[0198] III. EDAC/NHS Coupling of Avidin-D to 175 nm Beads

[0199] 4.4 mg sulfo-NHS was dissolved in 0.4 mL of a suspension of 25mg/mL carboxylate-modified 175 nm polystyrene beads (dyed with2-(p-dimethylaminophenyl)-3-phenyl dioxene/Eu(TTA)/TOPO) in water.0.0160 mL 0.25 M Na₂HPO₄ was added. 8 mg EDAC, dissolved in 0.030 mLwater, was added slowly to vortexing bead suspension. The suspension wasincubated for 20 min at room temperature. The beads were centrifuged 20min at 15,000 rpm in Sorvall SA-600 rotor at 4° C. The supernatant wasdiscarded. The beads were resuspended in 0:6 mL 0.005 M sodiumphosphate, pH 5.8. The suspension was sonicated to resuspend beads. Thebeads were again slowly added to 3 mL of a stirring solution containing1.33 mg/mL avidin-D (Vector) and 17 mM borax, pH 9.2, and mixed gentlyovernight at 4° C. 0.004 mL 1 M succinic anhydride in DMF was added. Thesuspension was incubated for 1 h at 4° C. with gentle mixing. 0.4 mL 50mg/mL BSA in 10 mM sodium phosphate and 150 mM NaCl, pH 7.0 was added.The suspension was allowed to mix gently for 3 h at 4° C. The beads werecentrifuged for 30 min at 15,000 rpm. The supernatant was discarded. Thebeads were resuspended in 3 mL 50 mM sodium phosphate and 150 mM NaCl,pH 7.6. The suspension was sonicated. The centrifugation, resuspensionand sonification steps were repeated for a total of three spins. Afterthe third spin, the beads were resuspended in 2.25 mL 50 mM sodiumphosphate and 150 mM NaCl, pH 7.6. The suspension was sonicated andstored at 4° C.

[0200] IV. Total T₃ Assay

[0201] Assay buffer: 0.075M barbital, 0.2M NaCl, 0.4% BSA, 1.25% mouseIgG, 10 mg/mL dextran sulfate (MW 500,000), 1.0 mg/mL dextran T-500, 10μg/mL aggregated IgG.

[0202] Beads

[0203] Acceptor Beads: Avidin-EDAC, 175 nm, dyed with2-(p-dimethylaminophenyl)-3-phenyl dioxene/Eu(TTA)₃/TOPO.

[0204] Sensitizer Beads: Antifluorescein-EDAC, 40 nm, dyed withchlorophyll-a/squarate.

[0205] Assay Protocol

[0206] 50 μL of 8-anilino-1-naphthalene sulfonic acid, ammonium salt(Sigma, A-3125) solution in assay buffer (0.75 mg/mL) was added to 50 μLof T₃ standard or sample. 100 μL of assay buffer was added. Biotinylatedanti-T₃ was prepared according to standard procedures by reaction ofbiotin-LC₇NHS (Pierce Chemical Company) with monoclonal anti-T₃ followedby purification by chromatography on a Sephadex column. 50 μL ofbiotinylated anti-T₃ (70 ng/mL) in assay buffer was added. The tracer,T₃-LC₂₁-Fl (1.8 ng/mL)

T₃-LC₂₁-Fl

[0207] in assay buffer (50 μL) was added. The mixture was incubated for15 minutes at 37° C. 500 μL of a suspension of sensitizer beads (50 μg)and acceptor beads (6.25 μg) in assay buffer were added, and the mixturewas incubated for 15 minutes at 37° C. The “stop solution” (50 μL) (10μM fluorescein, 0.5 mM biotin) was added.

[0208] Signal was read by halogen lamp with a 610 nm cut-off filter, oneminute illumination, 20 sec measurement.

[0209] Results

[0210] The luminescence signal was plotted as a function of T₃concentration. Signal modulation was 94% with 8.5 ng/mL T₃. At 0.5 ng/mLthe signal modulation was 38%.

Example 2 Chemiluminescence Quantum Yield and Decay Rate Determinations

[0211] Preparation of Compound 11

[0212] To a stirred solution of 2.55 g of 4-dimethylaminobenzoin (10mmol) in 50 mL of dry toluene, 1.2 mL of 2-mercaptoethanol (15 mmol) wasadded, followed by 2.5 mL of TMSCl. The reaction mixture was refluxedunder argon for 18 hours, allowed to come to room temperature and pouredin 150 mL of saturated bicarbonate solution. The two-phase mixture wasseparated. The organic layer was again washed with 100 mL of saturatedbicarbonate solution. The combined aqueous layer was extracted with 75mL of CH₂CL₂. The combined organic layers were dried over sodium sulfate(20 g) and evaporated. The remaining residue was flash chromatographed(CH₂Cl₂) to give 2.6 g of Compounds 11 and 20 (4:1 mixture of the2-regioisomers). The ash colored solid was recrystallized fromCH₂Cl₂-MeOH (10:90) mixture to yield 1.8 g of needle-shaped crystals ofa single regioisomer of compound 11.

[0213] M.P. 108-110° C.

[0214]¹HNMR (CDCl₃, 250 MHz): δ 2.85 (s, 6H), 3.22 (t, 2H), 4.5 (t, 2H),6.55 (d, 2H), 7.1-7.3 (m, 7H).

[0215] Mass Spectrum (CI: m/e, relative intensity) Major Peaks: 297 (M⁺,40), 165 (100).

[0216] Absorption Spectra (Toluene): 330 nm (ε 13,000).

[0217] Photooxygenation Procedure

[0218] 25 Milligrams of Compound 11 (major regioisomer from above) wasdissolved in 10 mL of CH₂Cl₂ in a photooxygenation tube. Approximately50 mg of polystyrene bound Rose Bengal was added and oxygen bubblerconnected. Oxygen was passed slowly through the solution while thesample as irradiated with a Dolan-Jenner lamp equipped with a 500 nmcut-off filter. Progress of the reaction was monitored by TLC. A spotfor the thioester product could be detected and had a lowerR_(f)(CH₂Cl₂) than Compound 11. The reaction was judged complete whenCompound 13 was completely consumed. The sensitizer was filtered off andsolution was evaporated on a rotary evaporator to yield 26 mg ofthioester 32 as the only product.

[0219] 1HNMR: (CD₂CL₂): δ 3.05 (s, 6H), 3.4 (5, 2H), 4.45 (5, 2H), 6.72(d, 2H), 7.5 (m, 3H), 7.85 (d, 2H), −8.05 (d, 2H).

[0220] Mass Spectra (CI, relative intensity) Major Peaks: 329 (M⁺, 25),148 (100).

[0221] Absorption Spectrum (CH₂Cl₂): 342 nm (−30,000)

[0222] Fluorescence Spectrum (Toluene): 370 nm.

[0223] Fluorescence Measurements

[0224] A solution of thioester 32 was taken in four different solvents(Toluene-dry; CH₂Cl₂; hexane; and acetonitrile) and placed in a 1-cmsquare quartz cuvette in the sample compartment of a Perkin-Elmer 650-40fluorometer. The sample was excited at the absorption maxima of eachsolvent (slit width 2 nm) and emission spectra (slit width 3 nm) wasrecorded by scanning from 350 nm to 470 nm. The fluorescence efficiencywas determined and tabulated in Table 1. TABLE 1 Efficiency of Thioesterin Different Solvents λ λ ABS EMI Compound Solvent nM nM Φ Diester*Toluene 314 360 0.1 400 Thioester 32** Toluene 338 370 0.025 CH₂Cl₂ 340390 0.07 Hexane 332 370 ˜0.006 CHCN 342 390 ˜0.006

[0225] Determination of Quantum Yield of Chemiluminescence.

[0226] Preparation of Eu(TTA)Phen:

[0227] 8.69 g of Eu(TTA)₃. 3H₂O(10 mmoles, Kodak) and 1.8 g of1,10-phenanthroline (10 mmoles, Aldrick) in 50 ml of dry toluene wereheated to 95° C. in an oil bath for one 1 hour. Toluene was removedunder reduced pressure. The ash coloured solid was cystallized from 10ml of toluene to yeild 10 grams of Eu(TTA)₃Phen.

[0228] Absorption spectrum: 270 nm (20,000), 340 nm (60,000) (Toluene)

[0229] 1.R(KBr): Cm⁻¹: 3440(s), 1600(s), 1540(s), 1400(s), 1300(s)

[0230] Energy Transfer to Eu(TTA)₃Phen

[0231] A solution of Compound 11 (regioisomers from above) (0.1 mM) (8:2mixture), aluminum phthalocyanine (0.1 μM), and Eu(TTA)₃Phen from above(0-4.0 mM) in dry toluene was placed in a 1-cm square quartz cuvette(two sided silvered) in the sample compartment of a Spex Fluorologspectrophotometer. The temperature of the sample holder was maintainedby a circulating external water bath at 25° C. A 640 nm cut-off filterwas placed in front of the excitation beam. The sample solutions wereplaced in the sample compartment for at least 3 minutes for thermalequilibrium to be reached. The emission was recorded in the time drivemode. Samples were irradiated at 680 nm (slit width 24 nm) until asteady state of emission at 613 nm (slit width 8 nm) was reached. Thesteady state light intensity at various concentrations of Eu(TTA)₃Phenwas recorded and is summarized in Table 2. From the steady state lightintensity quantum yields were determined. Double reciprocal plots ofchemiluminescence intensity against Eu(TTA)₃Phen concentration werelinear. TABLE 2 Chemiluminescence Efficiency as a Function of Eu(TTA)₃Phen Concentration Compound 11* Eu (TTA₃Phen mM mM RLU at 613 nm0.1 0 — 0.1 0.05 7.43 × 10⁴ 0.1 0.1  1.8 × 10⁵ 0.1 0.2 2.89 × 10⁵ 0.10.5 6.13 × 10⁵ 0.1 1.0 9.45 × 10⁵ 0.1 2.0 1.17 × 10⁶ 0.1 4.0 1.32 × 10⁶ 0.1** 4.0  1.6 × 10⁶

[0232] Chemiluminescence from Dioxene 9

[0233] Experiment 1: A solution of dioxene 9 (0.1 mM) and aluminumphthalocyanine (0.1 μM) in dry toluene was irradiated at 680 nm asdescribed above. The emission in light intensity at 400 nm (slit width 8nm) was recorded as a function of irradiation time. The light intensitywas 8793 RLU's for 180 seconds of irradiation (average of threeexperiments).

[0234] Experiment 2: Rate of dioxene 9 dioxetane decomposition wasmonitored by decay of chemiluminescence of an aerated solution in drytoluene at 0.25° C. Rate of decomposition was monitored in the presenceof 1.0 μM aluminum phthalocyanine and dioxene (less than 0.1 mM ofdioxene). The chemiluminescence decay was monitored on Spex Flubrologspectrophotometer under previously described conditions. The rateconstant of decay at 25° C. was 2.88×10⁻⁴ S^(−1.)

[0235] Preparation of Acceptor Beads

[0236] Four mL of 20% suspension (400 mg) of washed 175 nm carboxylatemodified latex was diluted with 3 mL of ethoxyethanol in a 25 mL roundbottom (R.B.) flask with a stir bar. The R.B. flask was then placed inan oil bath at 105° C. and stirred for 10 minutes. Then, 3.3 mM thioxene11 and 15.5 mM Eu(TTA)₃DPP was added; the beads were stirred for 5minutes more. At this point 1.0 ml of 0.1N NaOH was added slowly over 5minutes. During all the additions, the oil bath temperature wasmaintained at 105° C. The oil bath temperature was slowly allowed todrop to room temperature over 2 hours.

[0237] After cooling, the mixture was diluted with 20 mL of ethanol andcentrifuged (12,500 rpm, 30 minutes). Supernatants were discarded andthe pellets resuspended in ethanol by sonication. Centrifugation wasrepeated, and the pellet was resuspended in water; and centrifugationwas repeated. The pellet was resuspended in 5 mL of aqueous ethanol to afinal volume of 40 mL. The final concentration of the beads was 10mg/mL.

[0238] The concentration of Eu(TTA)₃DPP was determinedspectrophotometrically. An aliquot of the bead suspension was reduced todryness under a stream of dry argon and the residue dissolved indioxane. Using a density of 1.06 g/cc for polystyrene, (ε 340nm=6.7×10⁴) for Eu(TTA)₃ and (ε 270 nm=4.0×10⁴) for DPP, theconcentration of Eu(TTA)₃DPP was determined to be 100 mM. Theconcentration of compound 11 in the beads could not be determinedbecause its absorbance was masked by Eu(TTA)₃DPP.

[0239] Chemiluminescence of the beads was measured in an ORIELluminometer using water-soluble aluminum phthalocyanine sensitizer. Analiquot of beads was diluted to 100 μg/mL in phosphate buffer pH 8.0containing 0.1% Tween-20. 1.0 μM of aluminum phthalocyaninetetrasulfonic acid was added and chemiluminescent signal was measured asa function of irradiation time. An identical sample was also placed in aSpex Fluorolog fluorometer and irradiated at 680 nm (slit width 20 nm;640 cut-off filter). The chemiluminescence emission spectra was recordedby scanning from 570 nm to 620 nm. Chemiluminescence decay and quantumyields is summarized in Table 3.

[0240] Determination of Quantum Yields in Beads

[0241] Dioxene 9 Beads

[0242] A solution of dioxene 9 beads (0.2 mg) and aluminumphthalocyanine tetrasulfonic acid (2.5 μM) in phosphate buffer (pH 8.2;50 mM 0.1% Tween-20) was placed in a 1 cm quart cuvette (two sidessilvered) in the sample compartment of a Spex Fluorologspectrophotometer. The temperature of the sample holder was maintained25° C. A 640 cut-off filter was placed in front of the excitation beam.The sample solutions were placed in the sample compartment for at least3 minutes for thermal equilibrium to be reached. The light emission at360 nm was followed in the time drive mode. Samples were irradiated at680 nm (slit width 24 nm) for 60 seconds. The emission at 360 nm (slitwidth 16 nm) was recorded with time for 5000 seconds. Total lightemitted was determined by the cut-weigh method. Peak shape correctionwas also done by the cut and weigh method. The total light emitted at360 nm was 8.87±0.2×10⁴ RLU's/4500 seconds (after peak shape correction;average of 2 experiments).

[0243] Dioxene 9: Eu(TTA)₃TOPO Beads

[0244] A solution of dioxene 9 Eu(TTA)₃TOPO beads (0.2 mg) and aluminumphthalocyanine tetrasulfonic acid (2.5 μM) in phosphate buffer (pH 8.2,50 mM 0.1% Tween-20) was placed in a 1-cm quartz cuvette (two sidessilvered) in the sample compartment of a Spex Fluorologspectrophotometer. The rest of the experiment was performed as describedfor dioxene 9 beads. The light emission from beads was followed at 613nm (slit width 16 nm).

[0245] Total light emitted was determined by the cut and weigh method.PMT correction was done as described previously in solution studies. Thetotal light emitted at 613 nm was 25.0±0.3×10⁵ RLU's/4500 seconds (afterPMT correction; average of 2 experiments)

[0246] Steady State Methods

[0247] Dioxene 9: Eu(TTA)₃TOPO Beads. A solution of dioxene 9:Eu(TTA)₃TOPO beads (0.5 mg) and aluminum phthalocyanine tetrasulfonicacid (0.05 μM) in phosphate buffer (pH 8.2; 50 mM 0.1 Tween-20) wasplaced in a 12-75 mM test tube in the sample compartment of an Orielchemiluminometer. The temperature of the sample holder is 37° C. A 610cut-off filter was placed in front of the excitation beam. The samplesolutions were placed in the sample compartment for at least 5 minutesfor thermal equilibrium to be reached. The sample was irradiated for30-second intervals followed by a 5-second read time until a steadystate of emission is reached. The average intensity at steady stateemission is 21,000±1000 RLU's (3 experiments).

[0248] Compound 11: Eu(TTA)₃DPP Beads. A solution of thioxene 11:Eu(TTA)₃DPP beads (0.5 mg) and aluminum phthalocyanine tetrasulfonicacid (0.05 μM) in phosphate buffer (pH 8.2, 50 mM., 0.1% Tween-20) wasplaced in 12-75 mM test tube in the sample compartment of an Orielchemiluminometer. The temperature of the sample holder is 37° C. A 610cut-off filter was placed in front of the excitation beam. The samplesolutions were placed in the sample compartment for at least 5 minutesfor thermal equilibrium to be reached. The sample was irradiated for6-second intervals followed by 3 seconds read time until a steady stateof emission was reached. The average intensity at steady state emissionis 32,000±1000 (3 experiments). TABLE 3 Chemiluminescent Properties ofThioxene 11 and Dioxene 9 λmax Compound Medium (CH · EM) t½ Φ 11 Toluene400 nM 2.1 sec   low*  (100 μM) 11 + Toluene 613 nM 1.8-2.1 sec  0.20*** Eu (TTA) ₃Phen (4 mM) 11 + CML beads 613 nM decay 0.46 Eu(TTA) multiphasic ₃DPP (initial t½ (100 mM) at 37° C. is −0.5 secs) 9Toluene 420 nM 3462 sec  0.015 (100 μM) ** CML beads 360 nM decay  0.008multiphasic 9 + CML beads 613 nM decay 0.31 Eu (TTA)₃ · (Major)multiphasic TOPO 400 nM (16 mM)

Example 3

[0249] Preparation of C-26 Thioxene (Compound 13):

[0250] A. 62 g of N-methyl aniline (0.5 mole) and 62 g of ethyl5-bromovalerate (0.3 mole) were heated to 100° C. in a sealed tube for16 hours. The reaction mixture was cooled to room temperature and pouredinto 100 ml of ethyl acetate. The ethyl acetate solution was washed with20% sodium hydroxide (3×100 ml). The aqueous layer was extracted with 50ml of ethyl acetate. The combined ethyl acetate solution was dried oversodium sulphate (50 g) and removed under reduced pressure. The residuewas distilled under high vacuum (130-137° C.) to yield 60 g of N-methylN-ethyl valerate aniline.

[0251]¹H NMR (CDCl₃, 250 MHz): δ1.3 (t, 3H), 1.65 (m, 4H), 2.3 (t, 2H),2.8 (s, 3H), 3.3 (t, 2H), 4.2 (q, 2H), 6.65 (d, 2H), 7.2 (m, 3H).

[0252] B. To a stirred solution of DMF (8.8 g) in an ice bath POCl₃(5.06 g) was added slowly. After the addition was complete, the reactionis stirred at 4° C. for 10 minutes. N-methyl N-ethyl valeroyl anilinefrom Part A above (3.76 g) was added and the reaction was heated to 100°C. for 1 hour. The reaction mixture was poured into ice and neutralizedwith 20% sodium hydroxide. The mixture was extracted with ethyl acetate(3×50 ml). The combined ethyl acetate solution was dried over sodiumsulphate (50 g) and removed under reduced pressure. The residue waspassed through silica gel (CH₂Cl₂→CH₂Cl₂:EtOAc 9:2).

[0253]¹H NMR (CDCl₃, 250 MHz): δ1.2 (t, 2H), 1.6 (m, 4H), 2.3 (t, 2H),2.9 (s, 3H) 3.3 (t, 2H), 4.1 (q, 2H), 6.6 (d, 2H), 7.6 (d, 2H), 9.7 (S,1H)

[0254] C. To a refluxing solution of 5.0 g of N-methylN-ethyl-ω-valeroyl p-formyl aniline from Part B above (20 mmole) and 2 gof potassium cyanide in 60% ethanol under argon was added 2.15 g ofbenzaldehyde. (20 mmole) in 20 ml of ethanol in 90 minutes. The reactionmixture was refluxed for 15 minutes more and extracted with ethylacetate (3×50 ml). The combined ethyl acetate solution was dried oversodium sulphate (50 g) and removed under reduced pressure. The productwas purified on preparative TLC (hexane:ethyl acetate 5:1) to yield 2.2g of substituted benzoin.

[0255]¹H NMR (CDCl₃, 250 MHz): δ1.3 (t, 3H), 1.6 (m, 4H), 2.4 (t, 2H),2.9 (s, 3H), 3.3 (t, 2H), 4.1 (q, 2H), 4.8 (d, 1H), 5.8 (d, 1H), 6.5 (d,2H), 7.3 (m, 5H), 7.8 (d, 2H).

[0256] D. To a stirred solution of the benzoin from Part C above (1.1 g)in 15 ml of ethanol was added 7 ml of water and 100 mgs of KOH. Thereaction was stirred at room temperature for 3 hours. TLC (silica gel,CH₂Cl₂:EtOAc 9:1) showed no starting material. The solvent wasneutralized and the carboxylic acid product was extracted with ethylacetate (5×50 ml). The combined ethyl acetate solution was dried oversodium sulphate. (50 g) and removed under reduced pressure. Thecarboxylic acid product was used as is for the next step.

[0257]¹H NMR (CDCl₃, 250 MHz): δ1.6 (m, 4H), 2.4 (t, 2H), 2.9 (s, 3H),3.3 (t, 2H), 5.8 (s, 1H), 6.5 (d, 2H), 7.3 (m, 5H), 7.8 (d, 2H).

[0258] E. To a stirred solution of the carboxylic acid from Part D above(1.7 g, 5 mmole) and didecyl amine (1.9 g, 6.3 mmole) in 80 ml of DMF at4° C. was added DPPA (1.8 g, 8 mmole) followed by addition of triethylamine (1.25 ml). The reaction mixture was stirred at 4° C. and then atroom temperature for 16 hours. The solvent was neutralized and theproduct was extracted with ethyl acetate (5×50 ml). The combined ethylacetate solution was dried over sodium sulphate (50 g) and removed underreduced pressure. The product was purified on preparative

[0259] TLC (CH₂Cl₂: ethyl acetate 9:1) to yield 2.6 g of substitutedamide benzoin.

[0260]¹H NMR (CDCl₃, 250 MHz): δ0.8 (t, 6H), 1.3 (m, 36H), 1.6 (m, 12H),2.3 (t, 2H), 2.7 (m, 4H), 3.0 (s, 3H), 3.3 (m, 6H), 4.8 (d, 1H), 5.8 (d,1H), 6.5 (d, 2H), 7.3 (m, 5H), 7.8 (d, 2H).

[0261] F. To a stirred solution of 1.5 g of substituted benzoin (2.5mmole) in 50 ml of dry toluene, 1.2 ml of 2-thioethanol (15 mmole) wasadded, followed by 2.5 ml of TMSCl. The reaction mixture was refluxed inan oil bath under argon for 30 hours. The reaction mixture was allowedto come to room temperature poured into 150 ml of saturated bicarbonatesolution. The organic layer was separated and washed with 100 ml ofsaturated bicarbonate solution. The combined aqueous layer was extractedwith 75 ml of CH₂Cl_(2.) The combined organic solution was dried oversodium sulphate (50 g) and removed under reduced pressure. The productwas purified on silica gel (CH₂Cl₂: ethyl acetate 9:1) to yield 1.2 g ofC-26 thioxene as an pale yellow oil.

[0262]¹H NMR (CDCl₃, 250 MHz): δ0.8 (t, 6H), 1.3 (m, 36H), 1.6 (m, 12H),2.3 (t, 2H), 2.8 (s, 3H), 3.3 (m, 9H), 4.5 (t, 2H), 6.5 (d, 2H) 7.1 (d,2H), 7.3 (m, 5H).

[0263] Mass Spectrum (CI: m/e) M⁺ 662

[0264] Absorption Spectra (Toluene): 330 nm (ε13,000).

Example 4

[0265] Preparation of C-8 Thioxene (Compound 15):

[0266] A. To a refluxing solution of 3.0 g ofp-dimethylamino-benzaldehyde (20 mmole) and 2 g of potassium cyanide in60% ethanol under argon was added 4.4 g of p-octyl benzaldehyde (20mmole, Kodak) in 20 ml of ethanol in 90 minutes. The reaction mixturewas refluxed for 15 minutes more and extracted with ethyl acetate (3×50ml). The combined ethyl acetate solution was dried over sodium sulphate(50 g) and removed under reduced pressure. The product was purified onpreparative TLC (hexane:ethyl acetate 5:1) to yield 1.2 g of substitutedbenzoin.

[0267]¹H NMR (CDCl₃, 250 MHz): δ0.85 (t, 3H), 1.3 (m, 12H), 1.5 (m, 2H),2.5 (t, 2H), 2.9 (s, 6H), 4.8 (d, 1H), 5.8 (d, 1H), 6.5 (d, 2H), 7.3 (q,4H), 7.8 (d, 2H).

[0268] B. To a stirred solution of 0.94 g of substituted benzoin fromPart A above (2.5 mmole) in 50 ml of dry toluene, 1.2 ml of2-thioethanol (15 mmole) was added, followed by 2.5 ml of TMSCl. Thereaction mixture was refluxed in an oil bath under argon for 30 hours.The reaction mixture was allowed to come to room temperature and pouredinto 150 ml of saturated bicarbonate solution. The organic layer wasseparated and washed with 100 ml of saturated bicarbonate solution. Thecombined aqueous layer was extracted with 75 nl of CH₂Cl₂. The combinedorganic solution was dried over sodium sulphate (50 g) and removed underreduced pressure. The product was purified on silica gel (CH₂Cl₂: ethylacetate 9:1) to yield 0.75 g of C-8 thioxene Compound 15 as pale yellowsolid.

[0269]¹H NMR (CDCl₃, 250 MHz): δ0.8 (t, 3H), 1.3 (m, 10H), 1.6 (m, 2H),2.5 (t, 2H), 2.9 (s, 6H), 3.3 (t, 2H), 4.5 (t, 2H), 6.5 (d, 2H), 7.1 (d,2H), 7.3 (m, 5H).

[0270] Mass Spectrum (CI: m/e, relative intensity) 409 (M⁺100), 165(40).

[0271] Absorption Spectra (Toluene): 330 nm (ε13,000).

Example 5

[0272] Preparation of N-Phenyl Oxazine (Compound 16):

[0273] A. 5 g of p-dimethylaminobenzoin was dissolved in 5 ml of CH₂Cl₂and stirred in an ice bath. 10 ml of SOCl₂ was added and the reactionmixture stirred for 1 hour. The solvent was removed under reducedpressure and the product was crystallized from MeOH.

[0274]¹H NMR (CDCl₃, 250 MHz): δ3.0 (s, 6H), 6.3 (s, 1H) 6.5 (d, 2H),7.4 (m, 5H) 7.8 (d, 2H).

[0275] B. 0.271 g of P-(2-phenyl-2-chloro acetyl) dimethylamino-benzene(1 mmole) and 0.274 g of N-(2 hydroxy ethyl) aniline (2.0 mmole) weredissolved in 3 ml of dry ethanol and heated in a sealed tube at 80° C.for 8 hours. On cooling the product crystallized out as pale yellowneedles., which was filtered and dried to yield 0.2 g of N-phenyloxazine Compound 15.

[0276] 1H NMR (CDCl₃, 250 MHz): δ3.0 (bs, 6H), 3.7 (bt, 2H), 4.4 (bt,2H), 6.5 (bd, 2H), 7.4 (m, 12H).

[0277] Mass Spectrum (CI: m/e, relative intensity) 356 (M⁺, 100), 180(70).

Example 6

[0278] Preparation of N-Phenyl Indole Oxazine (Compound 17):

[0279] 0.283 g of 3-(2-phenyl-2-chloro acetyl) N-methylindole (1 mmole)(H. Nakamura and T. Goto, Heterocyles, 10, 167-170 (1978) and 0.274 g of2-anilino ethanol (2.0 mmole) were dissolved in 3 ml of dry ethanol andheated in a sealed tube at 80° C. for 8 hours. On cooling the productcrystallized out as pale yellow needles which was filtered and dried toyield 0.21 g of N-phenyl indole oxazine Compound 17.

[0280] 1H NMR (CDCl₃, 250 MHz): δ3.7 (bs, 3H), 3.8 (bt, 2H), 4.4 (bt,2H), 7.2 (bm, 15H).

[0281] Mass Spectrum. (CI: m/e, relative intensity) 366 (M⁺100), 180(70).

Example 7

[0282] Table 4 summarizes the properties of Compounds 11, 16, and 17determined in a manner similar to that described in Example 2. TABLE 4Properties of Chemiluminescent Compounds And Compositions λmax λmax λmaxCompound** (AbS) (EMI) (CH.EM) t½ Φ 11 330 nM 400 nM 400 nM 2.1 sec Low*(b) 11 + Eu(TTA)₃ 615 nM 1.3 sec 0.0024 (a) (b) 11 + 615 nM 1.8 sec 0.14Eu(TTA)₃Phen (a) (b) 16 400 nM 550 nM 120 sec Low* (b) 16 + Eu(TTA)₃ 615nM 11 sec 0.005 (1.5 × 10⁻⁴ M) 16 + Eu(TTA)₃ 615 nM 3.5 sec 0.04 (5.0 ×10⁻⁴ M) (b) (c) (d) 17 550 nM 120 sec Low* 17 + Eu(TTA)₃ 615 nM 12 sec0.04 (0.6 × 10⁻⁴ M) 17 + Eu(TTA)₃ 615 nM 2 sec 0.026 (0.6 × 10⁻⁴ M) (b)(c) (d)

[0283] The above discussion includes certain theories as to mechanismsinvolved in the present invention. These theories should not beconstrued to limit the present invention in any way, since it has beendemonstrated that the present invention achieves the results described.

[0284] The above description and examples disclose the inventionincluding certain preferred embodiments thereof. Modifications of themethods described that are obvious to those of ordinary skill in the artare intended to be within the scope of the following claims and includedwithin the metes and bounds of the invention.

What is claimed is:
 1. A composition comprising: (a) a metal chelatecomprising a metal selected from the group consisting of europium,terbium, dysprosium, samarium, osmium and ruthenium in at least ahexacoordinated state and (b) a compound having a structural portionthat is a double bond substituted with two aryl groups, wherein one ofthe aryl groups is electron donating with respect to the other, anoxygen atom and an atom selected from the group consisting of oxygen,sulfur and nitrogen.
 2. The composition of claim 1 wherein said compoundhas the structural portion:

wherein X is O, S or N wherein the valency of N is completed withhydrogen or an organic radical consisting of atoms selected from thegroup consisting of C, O, N. S, and P and Ar and Ar′ are independentlyaryl wherein one of Ar or Ar′ is electron donating with respect to theother.
 3. A composition comprising a latex particulate material havingincorporated therein the composition of claim
 1. 4. The composition ofclaim 1 wherein Ar is selected from the group consisting of 5-member and6-member aromatic and heteroaromatic rings.
 5. The composition of claim1 wherein Ar is phenyl substituted with an electron donating group at aposition of the phenyl that is meta or para to the carbon that is bondedto the double bond and Ar′ is phenyl.
 6. A method for determining ananalyte which comprises: (a) providing in combination (1) a mediumsuspected of containing an analyte, (2) a photosensitizer capable in itsexcited state of activating oxygen to a singlet state, saidphotosensitizer associated with a specific binding pair (sbp) member,and (3) the composition of claim 3 wherein said particulate material hasbound thereto an sbp member, (b) treating said combination with light toexcite said photosensitizer, and (c) examining said combination for theamount of luminescence emitted therefrom, the amount of saidluminescence being related to the amount of analyte in said medium.
 7. Akit comprising in packaged combination: (a) the composition of claim 3and (b) a photosensitizer that is not in said composition and is capablein its excited state of activating oxygen to its singlet state.
 8. Thecomposition of claim 1 wherein said compound is:

wherein X′ is S or NR′ wherein R′ is alkyl or aryl, and D and D′ areindependently selected from the group consisting of alkyl and alkylradical.
 9. The composition of claim 8 wherein D and D′ are methyl. 10.The composition of claim 8 wherein R′ is lower alkyl.
 11. Thecomposition of claim 8 wherein X′ is S and D and D′ are alkyl.
 12. Thecomposition of claim 8 wherein X′ is N(CH₃) and D and D′ are alkyl. 13.A composition comprising: (a) a metal chelate comprising a metalselected from the group consisting of europium, terbium, dysprosium,samarium, osmium and ruthenium in at least a hexacoordinated state and(b) the compound of claim
 8. 14. A compound of the formula:

wherein X′ is S or NR′ wherein R′ is alkyl or aryl and D and D′ areindependently selected form the group consisting of alkyl and alkylradical.
 15. The compound of claim 14 wherein R′ is methyl or phenyl.16. A composition comprising: (a) a metal chelate comprising a metalselected from the group consisting of europium, terbium, dysprosium,samarium, osmium and ruthenium in at least a hexacoordinated state and(b) the compound of claim
 14. 17. A composition comprising a latexhaving incorporated therein a compound of the formula:

wherein X″ is O, S or NR″ wherein R″ is alkyl or aryl, n is 1 to 4, andAr and Ar′ are independently aryl wherein one of Ar or Ar′ is electrondonating with respect to the other and Y is hydrogen or an organicradical consisting of atoms selected from the group consisting of C, O,N, S, and P and m is 0 to
 2. 18. The composition of claim 17 wherein R″is methyl or phenyl.
 19. The composition of claim 17 wherein n is
 2. 20.The composition of claim 17 wherein Ar is selected from the groupconsisting of 5-member and 6-member aromatic and heteroaromatic rings.21. The composition of claim 17 wherein Ar is phenyl substituted with anelectron donating group at a position of the phenyl that is meta or parato the carbon that is bonded to the double bond and Ar′ is phenyl. 22.The composition of claim 17 comprising a metal chelate wherein saidmetal is selected from the group consisting of europium, terbium,dysprosium, samarium, osmium and ruthenium in at least a hexacoordinatedstate.
 23. A composition comprising a latex having incorporated thereina compound of the formula:

wherein X′ is S or NR′ wherein R′ is alkyl or aryl and D and D′ areindependently selected from the group consisting of alkyl and alkylradical.
 24. The compound of claim 23 wherein R′ is methyl or phenyl.25. A method for determining an analyte which comprises: (a) providingin combination (1) a medium suspected of containing an analyte, (2) aphotosensitizer capable in its excited state of activating oxygen to asinglet state, said photosensitizer associated with a specific bindingpair (sbp) member, and (3) a suspendible latex particulate materialcomprising a chemiluminescent compound, said particulate material havingbound thereto an sbp member, said chemiluminescent compound having theformula:

wherein X″ is O, S or NR″ wherein R″ is alkyl or aryl, n is 1 to 4, andAr and Ar′ are independently aryl wherein one of Ar or Ar′ is electrondonating with respect to the other and Y is hydrogen or an organicradical consisting of atoms selected from the group consisting of C, O,N, S, and P and m is 0 to 2, (b) treating said combination with light toexcite said photosensitizer, and (c) examining said combination for theamount of luminescence emitted therefrom, the amount of saidluminescence being related to the amount of analyte in said medium. 26.The method of claim 25 wherein said photosensitizer is incorporated in asecond suspendible particulate material.
 27. The method of claim 25wherein R″ is methyl or phenyl.
 28. The method of claim 25 wherein n is2 and Ar is selected from the group consisting of 5-member and 6-memberaromatic and heteroaromatic rings.
 29. The method of claim 25 wherein Aris phenyl substituted with an electron donating group at a position ofthe phenyl that is meta or para to the carbon that is bonded to thedouble bond and Ar′ is phenyl.
 30. The method of claim 25 wherein saidphotosensitizer is a dye capable in the excited state of activatingmolecular oxygen to singlet oxygen.
 31. The method of claim 30 whereinsaid dye is selected from the group consisting of methylene blue, rosebengal, porphyrins, and phthalocyanines.
 32. The method of claim 25wherein said sbp members are independently selected from the groupconsisting of receptors, ligands, and polynucleotides.
 33. The method ofclaim 25 wherein said analyte is selected from the group consisting ofdrugs, proteins, nucleic acids and microorganisms.
 34. The method ofclaim 25 wherein said method is a homogenous immunoassay.
 35. The methodof claim 25 wherein said combination is treated by irradiation to excitesaid photosensitizer.
 36. The method of claim 25 wherein saidcombination is irradiated with light having a wavelength of 450-950 nm.37. The method of claim 25 wherein said sbp member associated with saidphotosensitizer is avidin or an antibody and said sbp member of saidchemiluminescent compound is avidin or an antibody.
 38. The method ofclaim 25 wherein said particulate material comprises a metal chelatecomprising a metal selected from the group consisting of europium,terbium, dysprosium, samarium, osmium and ruthenium.
 39. A method fordetermining an analyte which comprises: (a) providing in combination (1)a medium suspected of containing an analyte, (2) a photosensitizercapable in its excited state of activating oxygen to a singlet state,said photosensitizer associated with a specific binding pair (sbp)member, and (3) a suspendible latex particulate material comprising achemiluminescent compound, said particulate material having boundthereto an sbp member, said chemiluminescent compound having theformula:

wherein X′ is S or NR′ wherein R′ is alkyl or aryl and D and D′ areindependently selected from the group consisting of alkyl and alkylradical. (b) treating said combination with light to excite saidphotosensitizer, and (c) examining said combination for the amount ofluminescence emitted therefrom, the amount of said luminescence beingrelated to the amount of analyte in said medium.
 40. The method of claim39 wherein R′ is methyl or phenyl.
 41. The method of claim 39 whereinsaid particulate material comprises a metal chelate comprising a metalselected from the group consisting of europium, terbium, dysprosium,samarium, osmium and ruthenium in at least a hexacoordinated state. 42.A kit comprising in packaged combination: (1) a composition comprising asuspendible latex particle comprising a chemiluminescent compound of theformula:

wherein X″ is O, S or NR″ wherein R″ is alkyl or aryl, n is 1 to 4, andAr and Ar′ are independently aryl wherein one of Ar or Ar′ is electrondonating with respect to the other and Y is hydrogen or an organicradical consisting of atoms selected from the group consisting of C, O,N, S, and P and m is 0 to 2, said particle having bound thereto aspecific binding pair (sbp) member, and (2) a photosensitizer capable inits excited state of activating oxygen to its singlet state.
 43. The kitof claim 42 comprising a composition comprising a second suspendibleparticle comprising said photosensitizer, said second particle havingbound thereto a sbp member.
 44. The method of claim 42 wherein saidparticulate material comprises a metal chelate comprising a metalselected from the group consisting of europium, terbium, dysprosium,samarium, osmium and ruthenium.